Structural alterations of the EGF receptor genes in human tumors

ABSTRACT

Deletions in the EGF-R gene are found in many gliomas, breast tumors, and lung tumors. A particular truncated EGFR protein has been found in many tumors and provides diagnostic and therapeutic modalities.

This application is a continuation of U.S. Ser. No. 09/264,723, filedMar. 9, 1999, now U.S. Pat. No. 6,127,126, which is a divisionalapplication of U.S. Ser. No. 08/479,808, filed Jun. 7, 1995, now U.S.Pat. No. 5, 981,725, which is a continuation-in-part of U.S. Ser. No.07/896,909, filed Jun. 11, 1992 now abandoned, which is a continuationof U.S. Ser. No. 07/531,410, filed Jun. 1, 1990 now abandoned, which isa continuation-in-part application of Ser. No. 07/404,226, filed Sep. 8,1989 now abandoned.

This invention was made with the support of the National Institutes ofHealth. The United States Government retains certain rights in theinvention.

FIELD OF THE INVENTION

The invention relates to tumors and carcinoma involving mutations of theepidermal growth factor receptor (EGFR).

BACKGROUND OF THE INVENTION

Tumor specific molecules to aid in better diagnosis and treatment ofhuman and animal cancer have been sought since the last century. Hardevidence of tumor-specific substances, based on molecular structuraldata, has been difficult to provide in most types of human cancer exceptthose based on virally-induced cancer and involving molecular structuresspecified by the virus genome. There have been extremely few examples oftumor-specific molecules based on novel molecular structures. In thecase of malignant human gliomas and other tumors potentially associatedwith amplification or changes in the epidermal growth factor receptormolecule, such as carcinoma of the breast and other human carcinomas,there have been no unequivocal demonstrations of structurally alteredmolecules with unique sequences.

The epidermal growth factor receptor (EGFR) is the 170 kilodaltonmembrane glycoprotein product of the proto-oncogene c-erb B. Thesequence of the EGFR gene is known (Ullrich et al., 1984). The EGFR geneis the cellular homolog of the erb B oncogene originally identified inavian erythroblastosis viruses (Downward et al., 1984; Ullrich, et al.1984). Activation of this oncogene by gene amplification has beenobserved in a variety of human tumors (Haley et al., 1987a), and inparticular, those of glial origin (Libermann et al., 1985; Wong et al.,1987; Yamazaki et al., 1988; Malden et al., 1988).

One major difference between v-erb B oncogenes and the normal EGFR geneis that the viral oncogenes are amino-truncated versions of the normalreceptor; they lack most of the extracytoplasmic domain but retain thetransmembrane and tyrosine kinase domains (Fung et al., 1984; Yamamotoet al., 1983, Nilsen et al., 1985; Gammett et al., 1986). This resultsin a protein that is unable to bind epidermal growth factor (EGF) butcan still phosphorylate other substrates (Gilmore et al., 1985; Kris etal., 1985), and has led to speculation that the v-erb B proteins areoncogenic because the kinase domain is unregulated and constitutivelyactive (Downward et al., 1984).

A variety of genetic alterations can occur in viral erb B oncogenes,e.g. amino acid substitutions and deletions in the carboxy terminus ofthe gene. Available evidence, however, argues that the amino truncationis critical to carcinogenesis. Amino truncations are a feature of allv-erb B oncogenes, including those that arise by promoter insertion orretroviral transduction (Nilsen et al., 1985; Gammett et al., 1986).

In contrast, carboxy-terminal deletions appear to be associated onlywith tumors that arise through retroviral transduction and seem todetermine host range and tumor type specificity (Gammett et al., 1986;Raines et al., 1985). Transfection experiments with amino-truncatedavian c-erb B genes or chimeric viral oncogene-human EGF receptorsdemonstrates that this deletion is sufficient alone to create atransforming protein (Pelley et al., 1988; Wells et al., 1988).

Amplification of the EGFR gene occurs in 40% of malignant human gliomas(Libermann et al., 1985; Wong et al., 1987), Rearrangement of thereceptor gene is evident in many of the tumors with gene amplification.The structural alterations seem to preferentially affect the aminoterminal half of the gene (Yamazaki et al., 1988; Malden et al., 1988),but the nature of the rearrangements has not been preciselycharacterized in any tumor.

Size variant EGFR genes and amplification have been reported in severalhuman cancers. (Humphrey et al., 1988; Bigner et al., 1988; Wong et al.,1987; and Humphrey et al., 1989) There has been no determination,however, of the molecular basis for the altered EGFR molecules in cells.A determination of the genetic changes responsible for these tumorswould present a significant step forward in the treatment and diagnosisof human carcinoma.

It would be desirable to have unique gene and peptide sequences forglioma EGFR. It would also be desirable to have a synthetic peptideagainst which monoclonal or polyclonal antibodies could be producedwhich demonstrate specificity against mutant EGFR.

Bibliography

Alitalo, K. (1984). Amplification of Cellular Oncogenes in Cancel Cells.Medical Biology 62:304-317.

Bartels, I., Grzeschik, K. H., Cooper, D. N., Schmidtke, J. (1986).Regional Mapping of Six Cloned DNA Sequences on Human Chromosome 7. Am.J. Hum. Genet. 38:280-287.

Bigner, S. H., Mark, J., Bullard, D. E., Mahaley, Jr., M. S., Bigner, D.D. (1986). Chromosomal Evolution in Malignant Human Gliomas Start withSpecific and Usually Numerical Deviations. Cancer Genet. Cytogenetics22:121-135.

Bigner et al., J. Neuropathol. Exp. Neurol., 47:191-205 (1988);

Bullard et al. (1986). In Vivo Imaging of Intracranial Human GliomaXenografts Comparing Specific with Nonspecific Radiolabeled MonoclonalAntibodies. J. Neurosurg. 64:257-262

Carpenter, G. (1987). Receptors for Epidermal Growth Factor and OtherPolypeptide Mitogens. Annual Review of Biochemistry 56:991-914.

Carrasquillo, et al., Cancer Treat. Rep., 68:317-328 (1984), “Diagnosisof and Therapy for Solid Tumors With Radiolabeled Antibodies and ImmuneFragments”.

Chomczynski, P., Sacchi, N. (1987). Single-step Method of RNA Isolationby Acid Guanidinium Thiocyanate-Phenol-Chloroform Extraction. Anal.Biochem 162:156-159.

Deininger, P. L. Jolly, D. J., Rubin, C. M., Friedman, T., Schmid, C. W.(1981). Base Sequence Studies of 300 Nucleotide Renatured Repeated HumanDNA Clones. Journal of Molecular Biology 151: 17-33.

Di Fiore, P. P., Pierce, J. H., Fleming, T. P., Hazan, R., Ullrich, A.,King, C. R., Schlessinger, J., Aaronson, S. A. (1987). Overexpression ofthe Human EGF Receptor Confers an EGF-Dependent Transformed Pheotype toNIH 3T3 Cells. Cell 51:1063-1070.

Downward, J., Yarden, Y., Mayes, E., Scarce, G., Totty, N., Stockwell,P., Ullrich, A., Schlessinger, J., Waterfield, M. D. (1984). CloseSimilarity of Epidermal Growth Factor Receptor and v-erb B OncogeneProtein Sequence. Nature 307:521-527.

European Patent Application 0 153 114 (1985)

Peinberg, A. P., Vogelstein, B. (1984). A Technique for RadiolabelingDNA Restriction Endonuclease Fragments to High Specific Activity. Anal.Biochem. 137:266-267.

Fung, Y. K., Lewis, W. G., Crittenden, L. B., Kung, H. J. (1984).Activation of the Cellular Oncogene c-erb B by LTR Insertion: MolecularBasis for Induction of Erythroblastosis by Avian Leukosis Virus. Cell33:357-368.

Gammett, D. C., Tracy, S. E., Robinson, H. L., (1986). Differences inSequences Encoding the Carboxy-Terminal Domain of the Epidermal GrowthFactor Receptor Correlate with Differences in the Disease Potential ofViral erbB Genes. Proc. Natl. Acad. Sci. USA 83:6053-6057.

Gilmore, T., DeClue, J. E., Martin, G. S. (1985). Protein Phosphorlytionat Tyrosine is Induced by the v-erb B Gene Product in Vivo and In Vitro.Cell 40:609-618.

Gubler, U., Hoffman, B. J., (1983). A Simple and Very Efficient Methodfor Generating cDNA Libraries. Gene 25:263-269.

Haley, J. D., Kinchington, D., Whittloe, N., Waterfield, M. D., Ullrich,A. (1987A). The Epidermal Growth Factor Receptor Gene in: Oncogenes,Genes, and Growth Factors Edited by: Guroff, G. 12th Edition. Chapter 2.pp. 40-76. Wiley, N.Y.

Haley, J., Whittle, N., Bennett, P., Kinchington, D., Ullrich, A.,Waterfield, M. (1987b). The Human EGF Receptor Gene: Structure of the110 kb Locus and Identification of Sequences Regulation itsTranscription. Oncogene Research 1:375-396.

Haley, J. D., Hsuan, J. J., and Waterfield, M. D. (1989). Analysis ofMammalian Fibroblast Transformation by Normal and Mutated Human EGFReceptors. Oncogene 4:273-283.

Henikof, S. (1984). Unidirectional Digestion with Exonuclease IIICreates Targeted Breakpoints for DNA Sequencing. Gene 29:351-359.

Humphrey, P. A., Wong, A. J., Vogelstein, B., Friedman, H. S., Wernerr,M. H., Bigner, D. D., Bigner, S. H. (1988). Amplification and Expressionof the Epidermal Growth Factor Receptor Gene in Human Glioma Xenografts.Cancer Research 48:2231-2238.

Humphrey et al., J. Neurooncol. (1989)

Hyrien, O., Debatisse, M., Buttin, G., de, Saint, Vincent, B. R. (1987).A Hotspot for Novel Amplification Joints in a Mosaic of Alu-like Repeatsand Palindromic A+ T-rich DNA. EMBO. J. 6:2401-2408.

Janson, C. H., Tehrani, M. J., Mellstedt, H., and Wigzell, H. (1989).Anti-idiotypic Monoclonal Antibody to a T-cell Chronic LymphaticLeukemia. Cancer Immunology Immunotherapy 28:22-232.

Kawasaki, E. S., Clark, S. S., Coyne, M. Y., Smith, S. D., Champlin, R.,Witte, O. N., McCormick, F. P. (1988). Diagnosis of Chronic Myeloid andAcute Lymphocytic Leukemias by Detection of Leukemia-Specific DNA mRNASequences Amplified in Vitro. Proc. Natl. Acad. Sci. USA. 85:5698-5702.

Kozak, M. (1987). An Analysis of 5′-Noncoding Sequences from 699Vertebrate Messenger RNAs. Nucleic. Acids. Res. 15:8125-8148.

Kris. R. M., Lax, I., Gullick, W., Waterfield, M. D., Ulirich, A.,Fridkin, M., Schlessinger, J. (1985). Antibodies Against a SyntheticPeptide as a Probe for the Kinase Activity of the Avian EGF Receptor andv-erB Protein. Cell 40:619-625.

Lax, I., Brugess, W. H., Bellot, F., Ullrich, A., Schlessinger, J.,Givol, D. (1988). Localization of a Major Receptor Binding Domain in theEpidermal Growth Factor by Affinity Labelling. Molecular and CellularBiology 8:1831-1834.

Lee et al. (1988). Therapeutic Efficacy of Antiglioma MesenchymalExtracellular Matrix ¹³¹I-Radiolabeled Murine Monoclonal Antibody in aHuman Glioma Xenograft Model. Cancer Research 48:539-566

Lehrman, M. A., Schneider, W. J., Sudhof, T. C., Brown, M. S.,Goldstein, J. L., Russell, D. W. (1985). Mutation in LDL Receptor:Alu-Alu Recombination Deletes Exons Encoding Transmembrane andCytoplasmic Domains. Science 227:140-146.

Libermann, T. A., Nusbaum, H. R., Razon, N., Kris, R., Lax, I., Soreq,H., Whittle, N., Waterfield, M. D., Ullrich, A., Schlessinger, J.(1985). Amplification, Enhanced Expression and Possible Rearrangement ofEGF Receptor Gene in Primary Human Brain Tumours of Glial Origin. Nature313:144-147.

Malden, L. T., Novak, U., Kaye, A. H., Burgess, A. W. (1988). SelectiveAmplification of the Cytoplasmic Domain of the Epidermal Growth FactorReceptor Gene in Glioblastoma Multiforme. Cancer Research4:2711-2714.

Meeker, et al., Blood, 65:1349-1363 (1985), “A Clinical Trial ofAnti-Idiotype Therapy for B Cell Malignancy”.

Merlino, G. T., Ishii, S., Whang, P. J., Knutsen, T., Xu, Y. H., Clark,A. J., Stratton, R. H., Wilson, R. K., Ma, D. P., Roe, B. A. et al.(1985). Structure and Localiztion of Genes Encoding Aberrant and NormalEpidermal Growth Factor Receptor RNAs from A431 Human Carcinoma Cells.Molecular Cellular Biology 1 1722-1734.

Nilsen, T. W., Maroney, P. A., Goodwin, R. Rottman, R. M., Crittenden,L. B., Raines, M. A. Kung, H. J. (1985). c-erbB Activation inALV-Induced Erythroblastosis: Novel RNA Processing and PromoterInsertion Results in Expression of an Amino-Truncated EGF Receptor. Cell41:719-726.

Nister, M., Libermann, T. A., Betsholtz, C., Petterrson, M.,Claesson-Welsh, L., Heldin, C-H., Schlessinger, J., Westermark, B.(1988). Expression of Messenger RNA's from Platelet-Derived GrowthFactor and Transforming Growth Factor a and their Receptors in HumanMalignant Glioma Cells. Cancer Research 48:3910-3918.

Pelley, R. J., Moscovici, C., Hughes, S., Kung, H. J. (1988).Proviral-Activated c-erbB is Leukemogenic but not Sarcomagenic:Characterization of a Replication—Competent Retrovirus Containing theActivated c-erbB. Journal of Virology 62: 1840-1844.

Raines, M. A., Lewis, W. G., Crittenden, L. B., Kung, H. J. (1985).c-erbB Activation in Avian Leukosis Virus-Induced Erythroblastosis:Clustered Integration Sites and the Arrangement of Provirus in thec-erbB Alleles. Proc. Natl. Acad. Sci. USA 82:2287-2291.

Reed, K. C., Mann, D. A. (1985). Rapid Transfer of DNA from Agarose Gelsto Nylon Membranes. Nucleic Acids Research 1:7207-7221.

Riedel, H., Massoglia, S., Schlessinger, J., Ullrich, A. (1988). LigandActivation of Overexpressed Epidermal Growth Factor Receptors TransformsNIH 3T3 Mouse Fibroblasts. Proc. Natl. Acad. Sci. USA 8: 1477-1481.

Ruppert, J. M., Kinzier, K. W., Wong, A. J., Bigner, S. H., Kao, F. T.,Law, M. L., Seuanez, H. B., O'Brien, S. J., Vogelstein, B. (1988). TheGLI-Kruppel Family of Human Genes. Molecular and Cellular Biology8:3104-3113.

Russel, M., Kidd, S., Kelly, M. R. (1986). An Improved FilamentousHelper Phage for Generating Single-Stranded Plasmid DNA. Gene45:333-338.

Sealey, P. G., Whittaker, P. A., Southern, E. M. (1985). Removal ofRepeated Sequences from Hybridization Probes. Nucleic Acids Res. 13:1905-1922.

Sears, et al., The Lancet, April 3, 1982, pp. 762-765, (1982) “Phase-IClinical Trial of Monoclonal Antibody in Treatment of GastrointestinalTumours”.

Sears, et al., J. Biol. Resp. Mod., 3:138-150 (1984), “Effects ofMonoclonal Antibody Immunotherapy on Patients With GastrointestinalAdenocarcinoma”.

Sears, et al., Cancer Res., 45:5910-5913 (1985), “Phase II ClinicalTrial of a Murine Monoclonal Antibody Cytotoxic for GastrointestinalAdenocarcinoma”.

Steck, P. A., Lee, P., Hung, M. C., Yung, W. K. A. (1988). Express of anAltered Epidermal Growth Factor Receptor by Human Glioblastoma Cells.Cancer Research 48:5433-5439.

Ullrich, A., Coussens, L., Hayflick, J. S., Dull, T. J., Gray, A., Tam,A. W., Lee, J., Yarden, Y., Libermann, T. A., Schlessinger, J., et al.(1984). Human Epidermal Growth Factor Receptor cDNA Sequence andAberrant Expression. of the Amplified Gene in A431 Epidermoid CarcinomaCells. Nature 309:418-425.

Velu, T. J., Beguinot, L., Vass, W. C., Willingham, M. C., Merlino, G.T., Pastan, I., Lowry, D. R. (1987). Epidermal Growth Factor DependentTransformation by a Human EGF Receptor Proto-Oncogene. Science238:1408-1410.

Vogelstein, B., Fearon, E. R., Hamilton, S. R., Preisinger, A. C.,Willard, H. F., Michelson, A.M., Riggs, A. D., Orkin, S. H. (1987).Clonal Analysis Using Recombinant DNA Probes from the X-Chromosome.Cancer Research 47:4806-4813.

Vogelstein, B. (1987). Rapid Purification of DNA from Agarose Gels byCentrifugation through a Disposable Plastic Column. Anal. Biochem.160:115-118.

Wells, A., Bishop, J. M. (1988). Genetic Determinants of NeoplasticTransformation by the Retroviral Oncogene v-erbB. Proc. Natl. Acad. Sci.USA 85:7597-7601.

Winship, P. R. (1989). An Improved Method for Directly Sequencing PCRAmplified Material using Dimethyl Suplhoxide. Nucleic Acids Research17:1266.

Winter, E., Yamamoto, F., Almoguera, C., Perucho, M. (1985). A Method toDetect and Characterize Point Mutations in Transcribed Genes:Amplification and Overexpression of the Mutant c-Ki-ras Allele in HumanTumor Cells. Proc. Natl. Acad. Sci. USA 82:7575-7579.

Wong, A. J., Bigner, S. H., Bigner, D. D., Kinzler, K. W., Hamilton, S.R., Vogelstein, B. (1987). Increased Expression of the Epidermal GrowthFactor Receptor Gene in Malignant Gliomas is Invariably Associated withGene Amplification. Proc. Natl. Acad. Sci. USA 84:6899-6903.

Xu, Y. H., Ishii, S., Clark, A. J., Sullivan, M., Wilson, R. K., Ma, D.P., Roe, B. A., Merlino, G. T., Pastan, I. (1984). Human EpidermalGrowth Factor Receptor cDNA is Homologous to a Variety of RNAsOverproduced in A431 Carcinoma Cells. Nature 309:806-810.

Yamamoto, G., Hihara, H., Nishida, T., Kawai, S., Toyashima, K. (1983).A New Avain Erythroblastosis Virus, AEV-H Carries erbB Gene Responsiblefor the Induction of Both Erythroblastosis and Sarcoma. Cell 34:225-232.

Yamazaki, H., Fukui, Y., Ueyama, Y., Tamaoki, N., Kawamoto, T.,Taniguchi, S., Shibuya, M. (1988). Amplification of the Structurally andFunctionally Altered Epidermal Growth Factor Receptor Gene (c-erbB) inHuman Brain Tumors. Molecular and Cellular Biology 8:1816-1820.

Fingerling, S., Tsui, L. C., Grzeschik, K. H., Olek, K., Riordan, J. R.,Buchwald, M. (1987). Mapping of DNA Markers Linked to the CysticFibrosis Locus on the Long Arm of Chromosome 7 [Published ErratumAppears in Am J Hum Genet 1987 Aug:41(2):330] Am. J. Hum. Genet.40:228-236.

SUMMARY OF THE INVENTION

It is an object of the invention to provide intron free DNA moleculesand peptides that correspond to mutant EGFR proteins.

It is another object of the invention to provide antibodies specific formutant EGFR molecules that exhibit little or no cross-reactivity withnormal human EGFR or other tissues.

It is a further object of the invention to provide diagnostic methodsfor the detection of tumors.

It is an additional object of the invention to provide methods for thetreatment of tumors.

In accordance with these and other objects, one aspect of the inventioncontemplates an intron-free DNA molecule which encodes an EGFR mutanttype I, II or III peptide.

In another aspect, the invention contemplates substantially pure EGFRmutant types I, II or III peptides. The invention also contemplatesantibodies which specifically react with the EGFR mutants but which donot cross-react with normal, intact EGFR. Still further aspects of theinvention relate to the diagnosis of tumors by determining the presenceof the mutant EGFR proteins or the genes coding for them.

In yet another aspect, the invention contemplates the treatment oftumors employing an antibody which is specific for EGFR mutant type I,II or III peptides.

The invention provides an important step forward in the diagnosis andtreatment of tumors associated with altered EGFR genes. These tumorshave previously been characterized by the presence of amplified EGFRgenes. The present discovery is based on the existence of specificdeletions/rearrangements in these amplified genes. These altered genesproduce mutant EGFR proteins that can be identified by specificantibodies. A variety of materials attached to the antibodies allowshighly specific diagnosis and treatment of tumors bearing thesedeletion/rearrangement sequences.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is an illustration of a mutant type I EGFR peptide and a nucleicacid sequence therefor.

FIG. 2 is an illustration of a mutant type II EGFR peptide and a nucleicacid sequence therefor.

FIG. 3 is an illustration of a mutant type III EGFR peptide and anucleic acid sequence therefor.

FIGS. 4A-4b and 4C-1-4C-4 show: an EcoRI map of the human EGFR gene; thestructures of mutant EGFR Types I-III genes; and Southern blothybridization results.

FIGS. 5A-E characterize the rearranged fragment of a Type m mutant EGFRgene.

FIGS. 6A-6C characterizes a Type III mutant EGFR transcript.

FIGS. 7A-7B and 7C-1-7C-3 show transcript analysis by RNase protection.

FIGS. 8A-C are analysis results of the gene products from mutant TypesI-III.

FIGS. 9 and 10 describe polyclonal antibodies raised against a Type IImutant EGFR peptide. FIG. 9 shows the reactivity of antisera from threerabbits with fusion junction peptide in an ELISA assay. Free peptide wasbound to polyvinyl chloride plates. FIG. 10, left, showsimmunoprecipitation of mutant EGFR protein but not intact EGFR byanti-peptide 2 antibody. On the right, immunoprecipitation by monoclonalantibody 528 of EGFR from A431-X, D-256 MG-X and D-270MG-X is shown.

FIGS. 11A-11F is the cDNA sequence of normal EGFR.

DETAILED DESCRIPTION OF THE INVENTION

One of the unexpected discoveries of this invention is that identicaldeletions (at the gene product level) were observed in tumors arising indifferent patients. Previous studies with glioma xenografts have shownthat protein expressed from the amplified EGFR gene is on the cellsurface (Humphrey et al., 1988). It is a finding of the presentinvention that there are amino acids present at some deletion junctionswhich are not present on normal cells. Thus, there are surface moleculesin these tumors against which tumor-specific antibodies can begenerated. Such tumor-specific antibodies can be used to diagnose andtreat these widespread gliomas. (See, for example, Janson et al. (1989)regarding the use of antibodies in the treatment of lymphoid leukemias.)

Mutant EGFR protein is present in cells exhibiting one or more of threetypes of genetic deletion and/or rearrangement which result in astructurally altered receptor. The mutations resulting in these threetypes of altered receptor are illustrated in FIGS. 1-3. The first classof deletions (Type I, FIG. 1) results in a gap in the extracytoplasmicdomain near the transmembrane domain. The second type of deletion TypeII, FIG. 2) results in the elimination of the distal portion of theextracytoplasmic domain of EGFR. The Type I and II in-frame deletionsproduce two new junction points in the amino acid sequence. A third typeof abnormality (Type III, FIG. 3) is characterized by a deletion of themajority of the external domain of the EGFR leaving substantially onlythe transmembrane portion and the intracytoplasmic domain.

Type III mutations leave little or no extracellular protein. Althoughantibodies may detect an extracellular sequence, it is preferable tosolubilize a cell with a Type m mutation in a detergent before exposureto antibodies.

The cDNA sequence corresponding to normal EGFR has been reported byUllrich, et al., in Nature, 309:418425 (1984). (FIG. 11) Intron-free DNAsequences encoding deletion mutant EGFR Types I, II and III can bedetermined from the information provided in FIGS. 1, 2 and 3respectively, in view of the previously disclosed sequence for thenormal receptor. In particular, a gene encoding deletion mutant EGFRType I contains DNA segments corresponding to base numbers 1817 and 2067of the normal sequence connected together as shown in FIG. 1 with thesegment corresponding to base numbers 1818-2066 deleted. A gene encodingdeletion mutant EGFR Type II contains DNA segments corresponding to basenumbers 1-274 and 1076 to end of the normal sequence connected togetheras shown in FIG. 2 with the segment corresponding to base numbers275-1075 deleted. A gene encoding deletion mutant EGFR Type III containsa DNA segment corresponding to base numbers 1817 to the end with thesegment corresponding to base numbers 1-1816 deleted. Other DNAsequences which encode the same amino acid sequences may be used togenerate EGFR mutant peptides in recombinant organisms, due to thedegeneracy of the genetic code. Intron-free DNA molecules may beobtained using reverse transcriptase, for example, and an EGFR mRNA as atemplate. Alternatively, the DNA molecules can be chemically synthesizedaccording to the sequences disclosed herein. Such molecules can beamplified using polymerase chain reaction (PCR) to facilitate analysisand manipulations.

The DNA sequences and DNA molecules of the present invention may beintroduced into a host cell by transformation or transfection (to createtransformed cells) and expressed using a wide variety of host/vectorcombinations. For example, useful vectors may comprise segments ofchromosomal, non-chromosomal (such as various known derivatives of SV40and known bacterial plasmids, e.g., plasmids from E. coli includingcolE1, pcR1 pBR322, pMB9 and RP4), or synthetic DNA sequences, phageDNAs (M13) including derivatives of phage (e.g., NM 989) and filamentoussingle-stranded DNA phages, vectors useful in yeasts (such as the 2uplasmid), vectors useful in eukaryotic cells (such as vectors useful inanimal cells, e.g. those containing SV-40 adenovirus and retrovirusderived DNA sequences) and vectors derived from combinations of plasmidsand phage DNAs (such as plasmids which have been modified to employphage DNA), or other derivatives thereof.

Such expression vectors are also characterized by at least oneexpression control sequence that may be operatively linked to the mutantEGFR DNA sequence inserted in the vector to control and regulate theexpression of the cloned DNA sequence. Examples of useful expressioncontrol sequences are the lac system, the tM system, the tac system, thetrc system, major operator and promoter regions of phage lambda, thecontrol region of fd coat protein, the glycolytic promoters of yeast(e.g., the promoter for 3-phosphoglycerate kinase), the promoters ofyeast acid phosphatase (e.g., Pho5), the promoters of the yeast a-matingfactors, and promoters derived from polyoma, adenovirus, retrovirus, orsimian virus (e.g., the early and late promoters of SV40), and othersequences known to control the expression of genes of prokaryotic oreukaryotic cells and their viruses or combinations thereof.

Furthermore, within each specific expression vector, various sites maybe selected for insertion of the DNA sequences of this invention. Thesesites are usually designated by the restriction endonuclease which cutsthem. They are well recognized by those of skill in the art. It is, ofcourse, to be understood that an expression vector useful in thisinvention need not have a restriction endonuclease site for insertion ofthe chosen DNA fragment. Instead, the vector can be joined to thefragment by alternative means. The host cell, expression vector, and inparticular the site chosen therein for insertion of a selected DNAfragment and its operative linking therein to an expression controlsequence, is determined by a variety of factors, e.g., number of sitessusceptible to a particular restriction enzyme, size of the protein andits susceptibility to proteolytic degradation by host cell enzymes,contamination or binding of the protein to be expressed by host cellproteins difficult to remove during purification; expressioncharacteristics, such as the location of start and stop codons relativeto the vector and an insertion site for a DNA sequence is determined bya balance of these factors, not all selections being equally effectivefor a given case.

Useful expression hosts may include well known eukaryotic andprokaryotic hosts, such as strains of E. coli, such as E. coli SG-936,E. coli HB 101, E. coli W3110, E. coli X1776, E. coli X2282, E. coliDHI, and E. coli MRC1, Pseudomonas, Bacillus, such as Bacillus subtilis,Streptomyces, yeasts and other fungi, animal cells, such as COS cellsand CHO cells, and human cells and plant cells in tissue culture.

Of course, not all host/expression vector combinations function withequal efficiency in expressing the DNA sequences of this invention or inproducing the polypeptides of this invention. However, a particularselection of a host/expression vector combination may be made by thoseskilled in the art. For example, the selection should be based on abalancing of a number of factors. These include compatibility of thehost and vector, toxicity of the proteins encoded by the DNA sequence tothe host, ease of recovery of the desired protein, expressioncharacteristics of the DNA sequences and the expression controlsequences operatively linked to them, biosafety, costs and the folding,form or any other necessary postexpression modifications of the desiredprotein.

One preferred means of obtaining the deletion mutant EGFR preparationsof this invention is to synthesize them according to standard techniquesknown in the art using the sequences taught herein. Another meanscontemplates culturing cells transfected with an expression vectorcomprising an intron-free DNA molecule corresponding to FIG. 1 or FIG. 2or FIG. 3 using culture conditions which are well-known in the art. Thecells are then harvested and the cell membrane fraction may be separatedby standard separation procedures, such as differential centrifugation,which are well-known in the art. A crude extract can be obtained bysolubilizing the cell membrane fraction with detergents.

Once a crude extract containing the deletion mutant EGFR is obtained,purification can be accomplished according to techniques which arewell-known in the protein purification art. For example, various typesof chromatography may be used. Columns which may be used include a DEAEcellulose column, or an anion exchange column, as well as a gelpermeation column.

The deletion mutant EGFR protein or peptide fragments thereof can alsobe purified using immunoaffinity techniques. As antibodies are providedhere which are specific for the epitopes shown in FIGS. 1, 2, and 3,peptides corresponding to Type I, II, or III mutants can be positivelyselected from a mixture of many proteins. The use of the antibodies ofthe present invention to purify the proteins of the invention allowsgood separation from those proteins which are most similar to them.Alternatively, peptides of this invention may be purified byimmunoaffinity using antibodies to the normal EGFR, especially toepitopes on the cytoplasmic domain. Of course, other techniques ofpurification are known in the art and can be used to purify the peptidesof the invention.

Those of ordinary skill in the art can select among the above techniquesto prepare the substantially pure peptides of this invention.Substantially pure mutant EGFR peptides within the contemplation of thisinvention are those which are substantially free of other humanproteins. Peptides according to the present invention are linearpolymers of amino acids which do not contain the full intact sequence ofEGFR found in normal cells. Typically these are greater than ten aminoacids in length and less than about fifty. Desirably the peptides arelong enough to elicit a unique antibody response but short enough sothat antibodies are not elicited which are immunoreactive with theintact EGFR protein.

The peptide product of the prokaryotic and eukaryotic hosts transformedwith the DNA sequences of this invention, can be employed in theproduction of antibodies.

The substantially pure preparation of polypeptide comprising the aminoacid sequence corresponding to the nucleotide sequences of FIGS. 1, 2and 3 can be made using any of the techniques which are known in theart. For example, the Merrifield technique (Journal of American ChemicalSociety, vol. 85, pp. 2149-2154, 1968), can be used. Substantial puritymeans that the preparation is greater than 75% free of other proteinsnormally found in human cells. Preferably the preparation is greaterthan 90% free of other human proteins. Polypeptides may be longer orshorter or have conservative amino acid changes which do not change theepitope(s) found on deletion mutant EGFR but not found on normal intactEGFR. Polypeptides can be tested to determine if they are able tostimulate mammals to produce antibodies which are immunoreactive withepitopes found on deletion mutant EGFR, but not found on normal EGFR.Methods of immunizing mammals to stimulate antibody production are wellknown in the art. Methods for testing the immunoreactivity of antibodiesfor known antigens are also well known.

The substantially pure preparation of polypeptide of the presentinvention can be used to affinity purify antibodies specific for thedeletion mutant EGFR protein. In addition, the preparation ofpolypeptide of the present invention can be used to stimulate productionof antibodies in a mammal by immunizing the mammal with the preparation.Such immunization may optionally employ coupling of the polypeptide to alarger immunogenic substance such as keyhole limpet hemocyanin. Foraffinity purification of antibodies, the polypeptide can be coupled toan inert matrix, such as agarose beads. Techniques for such coupling arewell known in the art. The preparation of the polypeptide can also beused to quantitate antibodies specific for deletion mutant EGFR in anantibody preparation. In such a case, the synthetic peptide will usuallybe coupled to a larger inert proteinaceous substance such as bovineserum albumin. Once again, the techniques for coupling polypeptides tosuch matrices are well known in the art.

As mentioned above, antibodies which are specific for the deletionmutant EGFR proteins in that they are immunoreactive with the deletionmutant EGFR protein but not with normal, intact EGFR can be made using asynthetic polypeptide of the present invention. Immunization of mammalssuch as rabbits, mice, goats, etc. to produce antibodies is well knownin the art. Such polyclonal antibody preparations can be purified usingimmunoaffinity techniques employing a synthetic polypeptide of thepresent invention. Such purification methods are well-known in the art.Monoclonal antibodies can also be raised which are specific for deletionmutant EGFR epitopes and do not cross-react with normal EGFR. Generally,a rat or mouse will be immunized with the synthetic polypeptide (ordeletion mutant protein) of the present invention and the rodent willlater be sacrificed and spleen cells recovered for fusion with myelomacells. Hybrid cells can be selected according to techniques known in theart, for example, selections involving complementation of twophenotypes, one from each parental cell. The antibody produced by eachhybrid cell clone can be screened individually to select antibodieswhich bind to epitopes on deletion mutant EGFR but not on normal, intactEGFR.

In order to screen for antibodies which are immunoreactive with epitopesfound on the deletion mutant EGFR gene product but not found on normalEGFR, a simple battery of tests can be performed. Antibodies can betested for immunoreactivity with deletion mutant EGFR using asubstantially pure prep aration of the protein, or with fragments of thedeletion mutant EGFR protein according to the present inventionconjugated to a larger moiety such as bovine serum albumin. The desiredspecific antibodies should be positive in either or both of these tests.The antibodies should also be tested for immunoreactivity with normal,intact EGFR; desired antibodies having absolute specificity for deletionmutant EGFR should be negative in such tests.

Antibodies can also be detected using this battery of tests which haverelative specificity for deletion mutant EGFR compared to normal EGFR.That is, some monoclonal antibodies can be found which react morestrongly with the deletion mutant protein than with the normal protein.These antibodies of relative specificity for mutant EGFR may also beuseful. Means for using them are discussed below.

Immunoaffinity techniques to purify monospecific polyclonal antibodiesreactive with deletion mutant EGFR but not with normal EGFR can be used.Similar binding properties are employed as in the tests described formonoclonal antibodies above. That is to say that antibodies whichimmunoreact with deletion mutant EGFR will be positively selected, whilethose that immunoreact with normal EGFR will be removed from the desiredantibody preparation.

Antibodies which show relative or preferential specificity for deletionmutant EGFR relative to normal EGFR can be rendered absolutely specificby altering the conditions under which immunoreactivity is assayed.Conditions in the assay medium which can be altered include, but are notlimited to: the ionic strength; the detergent concentration; theconcentration of chaotropic agents, such as urea, guanidine, andpotassium thiocyanate; and the pH. Alteration of these conditions leadsto destabilization of the various bonding forces which contribute toantibody-antigen binding. Titration of reagents altering each of theseconditions allows determination of a set of conditions where relativelyor preferentially specific antibodies immunoreact with deletion mutantEGFR but not with normal EGFR. Suitable ranges in which to vary thedestabilizing agent concentrations can readily be determined. Forexample, in order to alter ionic strength, potassium chloride can betitrated from about 0.05M to 2M. Detergents, either ionic or non-ionic,can be titrated from about 0.05% to 2%. Chaotropic agents can betitrated from about 0.5M to 8M. The range of pH can be titrated fromabout 2 to 10. Such conditions can be useful both to screen formonoclonal antibodies immunoreactive with deletion mutant EGFR and toassay for deletion mutant EGFR in various biological sources.

Diagnostically, knowing the EGFR lesions correlated with tumors of TypesI, II, and III permits the development of gene or antibody probes foraccurately diagnosing and classifying tumors.

The nucleotide sequences provided by the invention can be used to formgene probes in accordance with any of the standard techniques. The DNAprobes contemplated for use in this invention may be derived from theDNA of cell lines grown in vitro or xenografts maintained in vivo whichcontain the DNA spanning the deletion site. The size of a DNA probe canvary from approximately 20 nucleotides to hundreds of nucleotides. TheDNA probe may be radiolabeled, labeled with a fluorescent material, orthe like. Procedures for the preparation and labeling of DNA probes arewell known in the art.

The diagnostic test employing a DNA probe will employ a cell sample froman individual who is being screened for the presence of a tumor,including but not limited to breast cancers, lung cancers, and gliomas.Other tumors which harbor EGFR mutations may also be tested. The samplewill be isolated from the suspect tissue. DNA is recovered from the cellemploying standard techniques well known to those skilled in the art.The DNA is then incubated with a probe under conditions where homologoussequences hybridize but sequences which diverge do not, andhybridization is thereafter detected. Hybridization to adeletion-specific probe indicates the presence of the deletion. Enzymessuch as S1 nuclease can be employed to remove portions of a DNA or RNAmolecule which do not hybridize. The size of the duplex nucleic acidwhich remains can be determined by standard means. Duplexes which aresmaller than the probe indicate a deletion, rearrangement, or othermismatch. Thus probes which are useful may be derived from intact aswell as mutant alleles.

Antibodies of the invention are capable of binding to the mutant EGFRproteins and not to the intact EGFR protein from normal cells. Theseantibodies also permit the use of imaging analysis with isotopes,conjugated antibodies, or other ligands. Examples of suitable imagingagents are ¹²⁵I, ¹²³I, ¹³¹I, or Indium-111 conjugated to the antibodiesspecific for deletion mutants of the EGFR.

The antibodies of the present invention can be used to detect deletionmutant EGFR epitopes in histological sections of glioma tissue as wellas in other solid tumors, such as breast cancer and lung cancer. Tissuesamples are preferably solubilized with detergent to release membraneproteins into solution prior to immunological detection. One can detectantibody binding to extracts of tissue sections by any detection meansknown in the art for example, radioimmunoassay, enzyme-linkedimmunoadsorbent assay, complement fixation, nephelometric assay,immunodiffusion or immunoelectrophoretic assay. Alternatively, theantibodies can be used as an immunohistochemical reagents to visualizeEGFR mutant proteins in tissue sections.

In addition, the antibodies of the invention can be administered to apatient for imaging analysis. For such purposes the antibodies aretypically conjugated to an imaging agent, such as ¹²³I, ¹³¹I, orIndium-111. A diagnostically effective amount of antibody is one whichallows the observer to distinguish between normal tissues and thosecontaining mutant type EGFR. Determination of such amounts is within theskill of the art.

A particularly useful stain for use in enzyme-linked antibody assaysemploys peroxidase, hydrogen peroxide and a chromogenic substance suchas aminoethyl carbazole. The peroxidase (a well known enzyme availablefrom many sources) can be coupled to the antibody specific for deletionmutant EGFR or merely complexed to it via one or more antibodies. Forexample, a goat anti-peroxidase antibody and a goat antibody specificfor deletion mutant EGFR can be complexed via an anti-goat IgG. Suchtechniques are well known in the art. Other chromogenic substances andenzymes may also be used.

Radio-labeled antibodies may be specific for deletion mutant EGFR orsecond antibodies immunoreactive with antibodies specific for deletionmutant EGFR. Again, such techniques are well known. The precisetechnique by which mutant EGFR is detected in glioma patients is notcritical to the invention. Biochemical or immunological techniques cannow be used which do not employ immunohistochemistry, although that is apreferred method of the present invention.

One particularly preferred method of detecting and/or quantitatingdeletion mutant EGFR protein in solubilized samples employs acompetitive assay. An antibody immunoreactive with an epitope found ondeletion mutant EGFR but not found on normal EGFR is attached to a solidsupport such as a polystyrene microtiter dish or nitrocellulose paper,using techniques known in the art. The solid support is then incubatedin the presence of the fluid to be analyzed under conditions whereantibody-antigen complexes form and are stable. Excess and unboundcomponents of the fluid are removed and the solid support is washed sothat antibody-antigen complexes are retained on the solid support. Afixed amount of a polypeptide containing an epitope found on deletionmutant EGFR but not found on normal EGFR, is then incubated with thesolid support. The polypeptide binds to an antibody immunoreactive withmutant EGFR which is attached to the solid support. The polypeptide hasbeen conjugated to a detectable moiety, such as biotin, peroxidase orradiolabel, by means well known in the art. Excess and unboundpolypeptide is removed and the solid support is washed, as above. Thedetectable moiety attached to the solid support is quantitated. Sincethe deletion mutant EGFR and the polypeptide have competed for the sameantibody binding sites, the solubilized mutant EGFR in the fluid to beanalyzed can be quantitated by its diminution of the binding of thepolypeptide to the solid support. Antibodies employed in this assay maybe immunoreactive with deletion mutant EGFR but not with normal EGFR.Alternatively, relatively specific antibodies may be used underconditions which destabilize immunoreactivity with normal EGFR.Polyclonal antibodies which contain an antibody species immunoreactivewith an epitope on deletion mutant EGFR but not on normal EGFR, may alsobe used.

According to one particularly preferred method, diagnosis of breastcancer or glioma is accomplished by determining the presence of anucleic acid molecule containing the sequenceCTG-GAG-GAA-AAG-AAA-GGT-AAT-TAT-GTG-GTG-ACA or its ribonucleotideequivalent in a cell suspected of being cancerous. The method may beperformed using a hybridization technique or a polymerase chainamplification technique.

According to another method of diagnosing breast cancer or glioma asample suspected of being cancerous or a person suspected of harboringsuch a cancer is tested for the presence of a protein comprising asequence LEU-LU-GLU-LYS-LYS-GLY-ASN-TYR-VAL-VAL-THR. An antibody may beused which is preferably immunoreactive with EGFR mutant protein type IIbut not immunoreactive with an intact EGFR protein. The method may beperformed by administering to a patient a diagnostically effectiveamount of such an antibody conjugated to an imaging agent.Alternatively, a Western blotting technique, an enzyme-linkedimmunosorbent assay, a radioimmunoassay, or an immunohistochemical assaycan be used to determine the presence of the mutant protein in a sample.

A preferred diagnostic method for determination of Type III tumorsinvolves differential detection of the intracytoplasmic andextracytoplasmic domains. This detection may be on the gene or peptidelevel. On the gene level, nucleotide probes specific for theintracytoplasmic domain (base numbers 2192-3720 on FIG. 11) and for theextracytoplasmic domain (base numbers 190-2122) are used. DNA extractedfrom Type III tumors will hybridize with the intracytoplasmic but notthe extracytoplasmic probes. Similarly, detergent solubilized membraneproteins from suspect tissue samples can be tested immunologically usingantibodies specific for epitopes found in the intracytoplasmic and theextracytoplasmic domains. The antibodies can be prepared by immunizingmammals with peptides expressed from the sequences corresponding tothese domains, as indicated above, and selecting those antibodiesspecific to each domain using techniques that are well known to thoseskilled in the art. The membrane protein fraction from Type m tumorswill react with antibodies to the intracytoplasmic domain of EGFR butnot with antibodies specific for most the extracytoplasmic domain. Theparticular procedures for gene probe assays and immunoassays will bewell-known to those skilled in the art. Similarly, antibodies specificfor Type III EGFR mutant proteins can be used. Such antibodies willreact with the epitopes which are not present on intact EGFR.

Treatment may be with radioactive isotopes including ¹³¹I [Bullard etal. (1986) and Lee et al. (1988)] or appropriate drugs also conjugatedto those antibodies. A number of treatment protocols employingmonoclonal and polyclonal antibodies have been developed in the art ofcancer therapy which are useful for the present invention. Each of theseprotocols depends on the specificity of the antibody as a targetingagent for their respective tumor antigen: 1) immune system effectorcells—either endogenous (Sears, 1984 & 1985, colorectal carcinoma;Meeker, 1985, B lymphocyte malignancy; Shouval, 1987, liver cancer) orisolated from the patient and reinjected; (Sears, 1982, and Douillard,1986, colorectal carcinoma) 2) cytotoxic drugs; (EPO 0153 144, ricin) or3) radioactive isotopes (Carrasquillo, 1984, directing ¹³¹I tometastatic melanoma). In each case, the role of the antibody is todirect the active agent to particular tumor cells whose surfaces carryantigens corresponding to the respective antibodies.

Treatment comprises administration to a cancer patient of an effectiveamount of an antibody specific for the mutant EGFR and unreactive withnormal EGFR, said antibodies optionally labelled with radioactiveelements or conjugated to cytotoxic drugs. See, U.S. Pat. Nos. 4,454,106and 4,472,509 which are expressly incorporated herein by reference. Theappropriate level of antibody for treatment can be determined for eachpatient using standard methods and no more than routine experimentation.

According to a preferred method of treating breast cancer or glioma anantibody coupled to a therapeutic agent is administered to a patientwith breast cancer or glioma. The antibody is specificallyimmunoreactive with an amino acid sequenceLEU-GLU-GLU-LYS-LYS-GLY-ASN-TYR-VAL-VAL-THR. According to anotherpreferred method of treating breast cancer or glioma an antibody isadministered to a patient having glioma or breast cancer. The antibodyis immunoreactive with an amino acid sequenceLEU-GLU-GLU-LYS-LYS-GLY-ASN-TYR-VAL-VAL-THR.

The following examples are included for illustrative purposes only andare not intended to limit the scope of the invention.

EXAMPLES

In the examples, we determined the effect of rearrangements on thestructure of the EGFR gene product in five human glial tumors. Thetarget of each of these alterations was the extracytoplasmic domain.Three types of altered transcripts were identified.

A. Source of Tumor Material

The xenografts used in the present study were derived from surgicalbiopsies of malignant human gliomas. Their establishment and karyotypeshave been described previously (Humphrey et al., 1988). Southernblotting experiments with EGFR cDNA probes have shown that five of thexenografts (D245MG, D256MG, D270MG, G298MG and D317MG) containrearranged and amplified EGFR genes. The same rearranged fragments weredetected in the original tumor biopsies (Humphrey et al., 1988).

Tumor xenograft D320MG exhibited amplified EGFR genes without anydetectable rearrangements. Xenografts D263 and D274 exhibited noamplification or rearrangement of the EGFR gene. Therefore, five of the8 glioma xenografts examined contained rearranged and amplified EGFRgenes which were all present in the initial tumor biopsy.

B. Cloning of the EGF Receptor Gene Demonstrates Internal Deletions inHuman Glial Tumors

To analyze the structure of the altered EGF receptor genes, a map of thegene was constructed. We generated a genomic phage library using DNAfrom D320MG. The human glial tumor xenograft has an approximatelyten-fold amplification of the EGFR gene but no detectable rearrangementson Southern blot hybridization. The library was screened with fragmentsfrom a 5.5 kb EGF receptor cDNA clone (Ulirich et al., 1984).

Forty-eight phage clones were obtained and used to assemble an EcoRI mapof the EGF receptor gene (FIG. 4A). The clones spanned the entire geneexcept for two small gaps. Southern blot hybridization (using genomicDNA digested with various enzymes) confirmed that the clones identifiedall EcoRI fragments within the gene (including those containing thegaps). The map deduced using these tumor-derived phage clones was notrearranged in comparison to normal genomic DNA.

A map of the EGF receptor gene has been published by Haley et al. (Haleyet al., 1987b). Our map agrees with Haley et al. except that the size ofsome of the fragments differ for unknown reasons. In addition, our mapincludes several small EcoRI fragments not included previously. Theextracytoplasmic and transmembrane domains are located at base numbers186-2121 and base numbers 2122-2191, respectively for EGFR genes (FIG.11).

Clones corresponding to the extracytoplasmic domain of the gene wereused as hybridization probes with Southern blots of EcoRI digested tumorDNA (FIG. 4C). Each genomic clone revealed a deletion and/or arearrangement in at least one of the five tumors in which we hadpreviously demonstrated rearrangements using EGFR cDNA probes (Humphreyet al., 1988).

Based on the information obtained from Southern blots probed withgenomic phage clones, the approximate areas of deletion within the EGFRgene in each tumor were deduced (FIG. 4B). While none of theserearrangements were identical at the genomic level, the deletions inD256MG and D298MG were around a central locus. Tumors whose genomescarry this central deletion are designated Type I. The deletions inD2709MG and D317MG centered around a locus at the 5′ end of the gene.Tumors whose genomes carry this end deletion are designated Type II. Thedeletion in D245 appeared to involve most of the extracytoplasmicportion of the gene. Tumors whose genomes carry this deletion aredesignated Type III.

C. Summary of EGFR Mutant Type Analysis

Phage clones 40 and 24 revealed deletions and rearrangements in Type IItumors D270 (FIG. 4C, lane C) and D317 (data not shown), but not in TypeI tumors D256MG or D298MG. Conversely, clones 26 (FIG. 4C) and 29 (datanot shown) demonstrated a rearrangement in tumors D256MG (FIG. 4C, LaneB) and D298MG (lane E) but not in tumors D270 or D317. The abnormallymigrating fragments were unlikely to be the result of restrictionfragmentation length polymorphisms (RFLPs) since new bands were alsoobserved when the tumor DNA was digested with several other enzymes.

Using EcoRI subfragments of phage clone 26, the 2.5 kb fragment in TypeI tumor D256 (FIG. 4C, phage clone 26, Lane B) was shown to be due to arearrangement affecting the 8.0 kb and 1.8 kb fragments (FIG. 4A). Thefact that the normal size 8.0 kb fragment was still present at increasedcopy number in tumor D256 could be due to the presence of the severalcopies of chromosome 7 which were detected cytogenetically in metaphasespreads from this xenograft (Bigner et al., 1986). Such numericalincreases in chromosome 7 are common in glial tumors. As an alternativeexplanation, some of the amplification units in D256 may beheterogeneous with some units containing the normal 8.0 kb fragment andothers containing the rearranged 2.5 kb fragment.

Phage clones ERG-9, 40, and 24, demonstrated no amplification in TypeIII tumor D245MG (FIG. 4C, lane A). However, the phage ERG 26 probe,which hybridized to seven EcoRI fragments present in normal DNA,revealed only two amplified fragments in this tumor: a normal size 8.0kb fragment and an abnormally migrating 1.7 kb fragment. These resultssuggested that the 5′ part of the EGF receptor gene was deleted inD245MG.

D. Detailed Analysis of Type III Rearrangement

To better understand the nature of this deletion/rearrangement in tumorD245MG, we cloned the 1.7 kb EcoRI fragment containing the presumptiverearrangement from the tumor xenograft (FIG. 4C, Phage clone 26, LaneA), and subsequently used this clone to isolate the sequences givingrise to the rearrangement. The three phage clones spanning the site ofrecombination are shown in FIG. 5A. The sequence marked “EGFR” isderived from the normal EGF receptor gene (Ullrich et al., 1984), the“D245” sequence is derived from the amplification unit of D245MG, andthe “TEG” sequence is derived from the normal locus (referred to as TEGfor Truncated EGF Receptor) which had recombined with the EGFR gene toproduce the rearrangement in D245MG. Cross hybridization and restrictionmapping indicated that the 1.7 kb EcoRI restriction fragment from theD245MG DNA was the product of a rearrangement between a 1.8 EcoRifragment from the EGFR locus and a 2.2 kb EcoRI fragment from the TEGlocus.

The three fragments were subcloned into plasmid vectors and partiallysequenced. The nucleotide sequences are presented in FIG. 5B anddiagrammed in FIG. 5C. Several features of the sequences were notable:(i) the rearrangement occurred within an intron upstream of the EGFreceptor exon corresponding to nucleotides 1818 to 1908 of the EGFR cDNAsequence; (ii) an 18 nucleotide A-T rich motif was repeated four timesin the vicinity of the breakpoint in the rearranged fragment, but onlyonce in the corresponding part of the TEG fragment; (iii) at the site ofrecombination, seven additional nucleotides were present which did notappear to be derived from either the TEG or EGF receptor loci; and (iv)Alu-type repeats were present in both the TEG and EGF receptor derivedfragments and one such repeat is present in the clone containing therearrangement.

To determine the normal chromosomal position of the TEG locus, wescreened human-mouse somatic cell hybrids with the TEG specific probe.This demonstrated that TEG, like the EGF receptor gene, was located onchromosome 7. Further hybridization to hybrids containing variousdeletions on chromosome 7 (Bartels et al., 1986; Zergerling et al.,1987), showed that both TEG and the EGF receptor were located in thesame subchromosomal region at 7pl2-7pl4 (FIG. 5D). Thus, therearrangement in D245 was intrachromosomal. The juxtaposition of Alusequences around the breakpoints (FIG. SC) suggested a model for theD245 recombination similar to one proposed for the LDL receptor (Lehrmanet al., 1985), wherein hairpin stem-loop structures composed of Alu-typerepeats mediate an intra-strand recombination event (FIG. SE). Thesignificance of the A-T rich repeat is unclear, but a hotspot ofrecombination in a mouse model of gene amplification occurred within thecontext of an A-T rich area and Alu-type repeats (Hyrien et al., 1987).

E. The protein produced in D245MG is similar to v-erb B

To determine the effect of the truncation on the gene product, we firstperformed Northern blot analysis using poly A+ selected RNA from thexenograft of D245MG. When probed with sequences derived from the 3′ halfof the EGF receptor cDNA, normal placenta showed the expected 10.0 and5.5 kb transcripts, but abnormally migrating bands of 9.0 and 4.8 kbwere obtained with RNA from tumor D245 (FIG. 6A). When the blot washybridized with a probe from the 5′ half of the EGF receptor cDNA, thesame 10.0 and 5.5 kb bands were detected in placenta, but no transcriptwas found in RNA from D245MG. This was consistent with the Southern blotand genomic cloning data which had suggested that a deletion of the 5′end of this gene had occurred.

To determine the nature of the aberrant transcript from D245 cells, weconstructed a cDNA library using poly A+ selected RNA from the D245xenograft. A combination of restriction endonuclease mapping and partialsequence analysis of 17 different cDNA clones selected with a probe forthe EGF receptor gene showed that they were identical to publishedsequences of EGF receptor mRNA from nucleotide 885 to the 3′ end of thecoding region The sequences 5′ to nucleotide 885 of the D245 cDNA clonewere not homologous to EGF receptor cDNA and were apparently derivedfrom the upstream locus. Search of a DNA database (Gen Bank, release52.0) revealed no significant homologies of this sequence to any knownsequence. Open reading frame (ORF) analysis upstream of nucleotide 885showed numerous stop codons in three reading frames. The single longopen reading frame began at D245 cDNA nucleotide 955 and continued inthe native reading frame of the EGF receptor. The nucleotidessurrounding the first methionine codon (GCCATGA) within this ORF were ingood agreement with the canonical initiator sequence (Kozak et al.,1987). Thus, it is likely that the translation product of this RNA hadan N-terminus corresponding to amino acid 543 of the EGF receptorprotein and that the open reading frame was preceded by a long 5′untranslated sequence. The predicted protein product of this mRNA wouldbe a truncated version of the normal receptor containing 644 aminoacids, a predicted molecular weight of 72 kd, and retention of 3N-linked glycosylation sites (Ullrich et al., 1984). Comparison of thepredicted amino acid sequence from the D245 EGF receptor gene with thatof v-erb B oncogenes (Fung et al., 1984; Yamamoto et al., 1983; Nilsenet al., 1985; Gammett et al., 1986) revealed that the protein product ofthe tumor is very similar (and in fact only 8 amino acids longer) toseveral avian retroviral gene products described previously.

F. Detailed Analysis of Type I and Type II Transcripts

We first employed RNase protection to analyze the transcripts from thefour other tumors with EGFR rearrangements. In addition to being able todetect deletions in mRNA, this technique can reveal a significantfraction of point mutations (Winter et al., 1985). The probes used toanalyze total RNA from these tumors are shown in FIG. 7A. Probe Icontained a 910 base pair fragment from the 5′ end of the cDNA. Ityielded a fragment of 910 bp from all RNA samples tested (FIG. 7C); inthe RNA samples from the tumors with rearrangements, the normal sizefragments were probably a result of transcription from the remainingnormal EGFR gene. In previous cytogenetic experiments, we have shownthat at least two copies of chromosome 7 remain in each of these fourtumors (Bigner et al., 1986). In tumors D270 (FIG. 7C, probe I, lane C)and D3217 (Lane F), the most intense fragment detected was approximately230 bp; these new fragments appeared to be expressed at a considerablyhigher level than the normal size 910 bp fragment, especially when thedifference in size was taken into account.

Probe II revealed 210-215 base, pair fragments in tumors D270 and D317as well as less intense 914 base pair fragment corresponding to thenormal transcript (FIG. 4C, probe II, lanes C and F). The cluster ofclosely migrating fragments (rather than a single band) was most likelydue to imperfect cleavage of the duplex; RNase protection experiments inwhich similar RNA probes were hybridized to cloned genomic fragmentsalso produced a cluster of fragments. The data obtained with probes Iand II were consistent with the hypothesis that the deletion in bothtumors D270 and D317 resulted in an approximately 800 base pair deletionnear the 5′ end of the transcript. While the genomic deletions were notidentical FIG. 4), it appeared that the two tumors had lost similar exonsequences.

Probe III demonstrated the normal 970 base pair fragments in D270 andD317 (FIG. 7C, probe III, lanes C and F), but 450 and 250 bp fragmentswere observed in D256 and D298 (Lane B and E), suggesting anapproximately 270 base pair deletion within the transcript of theselatter two tumors. Tumor D245 also had an abnormal RNase protectionpattern (FIG. 7C, probe III, lane A); the 495 bp fragment in D245corresponded to the break point in the transcript determined from thecDNA clones. Probes specific for the 3′ half of the receptor (includingthe tyrosine kinase domain and autophosphorylation sites) showed noabnormalities in any of the five tumors, indicating that there were notpoint mutations detectable by this method.

G. The Rearrangements Result in In-Frame Deletions

The RNase protection and genomic Southern blot data provided anapproximation of the nature of the deletion, but it was difficult todetermine the precise nature of the abnormalities. For this purpose, thepolymerase chain reaction (PCR) was used to generate cDNA fragments fromthe mutant alleles. The data from the RNase protection experiments andpublished information on the normal EGFR cDNA sequence was used to guidethe choice of primers. cDNA was generated by first annealing the 3′primer to total tumor RNA, and then extended by using MMLV reversetrahscriptase. PCR was then carried out using a method similar to thatdescribed by Kawasaki et al. (Kawasaki et al., 1988). When RNA fromtumors D270 and D317 was analyzed with one set of primers (FIG. 8A) amajor fragment of 230 base pairs were produced (FIG. 8A, lanes A and B),while the normal size 1100 base pair fragment was seen only faintly (notshown), consistent with the size of the deletions inferred by the RNaseprotection experiments. Another glial tumor xenograft, D397, had aSouthern blot pattern very similar to that of D270 when probed with theEGFR cDNA. It was analyzed in this PCR experiment and produced afragment similar to that from D270 and D317 (FIG. 8A, lane C). A secondset of primers revealed a fragment of 220 base pairs in tumors D256 andD298 (FIG. 8A, lanes D and E respectively), instead of the normal size440 base pairs which was found with D320 RNA (lane F); the 220 bpfragment size was consistent with the data from the RNase protectionexperiments.

PCR products of the four tumors were subcloned and sequenced. Thesequences from tumors D270, D317 and D397 were all identical (FIG. 8B),and the sequence derived from tumor D256 was identical to that from D298(FIG. 8C). In all cases the deletions did not alter the reading frameand reconstituted a glycine codon at the deletion site.

Discussion

We have demonstrated that rearrangements of the EGFR gene in human glialtumors often result in specific deletions of the extracytoplasmic domainof the molecule. While coding information was lost from this domain, thereading frame of the receptor was precisely preserved in every instance.Our results confirm and extend the observations made by other workersusing Southern blot assays on glial tumors. Libermann et al. (Libermannet al., 1985) noted that two glial tumors with DNA amplification hadapparent rearrangements that were detected with cDNA probes from theextracytoplasmic domain. Similarly, Yamazaki and co-workers (Yamazaki etal., 1988) and Maiden et al. (Maiden et al., 1988) detectedrearrangements in two glial tumors and found that these produce proteinproducts of abnormally small size. Recently, a 190 Kd protein has beenisolated from a glial tumor cell line which did not appear to be theresult of any gross genetic rearrangement, but peptide mapping suggestedthat the additional material was due to an addition in theextracytoplasmic domain (Stech et al., 1988).

In a previous publication, we examined the size of the proteins producedin these xenografts and their effect on EGF binding affinity (Humphreyet al., 1988). As would be predicted from the sequence data presentedhere, the protein isolated from D245 is unable to bind EGF but can beautophosphorylated. The major polypeptide detected with antibodiesagainst the intracytoplasmic domain of the receptor was 77 kilodaltonsfollowing deglycosylation, consistent with the 72 kilodaltons predictedfrom the cDNA sequence. The proteins immunoprecipitated from tumors 270and 317MG were 150 kilodaltons (predicted polypeptide size of 120,000)and those from D298 and D256 were 130 kilodaltons (predicted polypeptidesize of 100,000). The differences in size from that predicted from thecDNA sequences may reflect the degree of glycosylation, as there is asimilar difference between the predicted molecular weight of normal EGFRprotein and its mobility on SDS gels. The binding affinity for EGF inthese tumors was less than three-fold lower than that of the controlcell lines with normal EGFR gene sequences. Thus, the deletions do notseem to substantially affect EGF binding, although the normal receptorsthat are still present in these tumors complicate this conclusion. Thedomain thought to be responsible for EGF binding (Lax et al., 1988) wasnot deleted in any of the tumors except for D245MG.

Several facts suggest that the alterations noted here play an importantrole in glial tumor development. First, in humans, gene amplificationonly appears in tumor cells (Alitalo et al., 1984), and with theexception of B and T lymphocytes, genetic rearrangements have only beenassociated with disease. Second, EGFR gene amplification in glial tumorsis a clonal event (i.e., amplified genes appeared to be present innearly every cell of the tumor (Wong et al., 1987). Third,amino-terminal truncations in v-erb B are known to be crucial for theireffect; in all of the tumors studied here, the alterations were confinedto the amino-terminal domain. In one tumor, the deletion resulted in atruncation remarkably similar to that found in v-erb B oncogenes.

When EGF binds to the normal receptors, autophosphorylation andactivation of the kinase domain occurs within minutes, and receptor downregulation through endocytosis and degradation of the ligand receptorcomplex are completed within one hour (Carpenter et al., 1987). Theproducts of the v-erb B oncogene are thought to be oncogenic because thetruncation results in a protein whose kinase activity is constitutivelyactive and unregulated (Downward et al., 1984). The structuralalterations occurring in D245MG may result in a similar activation ofthe receptor kinase, allowing it to function in the absence of thenatural ligand. It has recently been demonstrated that over-expressionof a protein lacking the EGF binding domain can transform Rat 1 cellsand activate the tyrpsine kinase (Haley et al., 1989). The deletions inthe other tumors studied occurred within one of the two cysteine richdomains in the receptor. Therefore, these alterations could also resultin a conformational change that might leave the receptor in anabnormally active or unregulated state.

Finally, one must ask why the rearrangements occur in amplified genes ofsome gliomas while other similar tumors contain amplified but otherwiseapparently normal genes. One possibility is that a certain subset ofglial tumors are dependent on the over-expression of EGF receptors.Evidence in support of this possibility comes from recent in vitroexperiments in which transformation of NIH 3T3 cells by over-expressionof the EGF receptor required the presence of EGF (Di Fiore et al., 1987;Riedel et al., 1988; Velu et al., 1987) and the demonstration that someglial cell lines contain TGF alpha transcripts (Nister et al., 1988).Extrapolating the in vitro data to the in vivo state, tumors possessinga high density of normal receptors might be activated by otherwiselimiting quantities of EGF or TGF-alpha present in the area surround andincluding the tumor.

A second subset of tumors might have altered receptors whose structureresults in a molecule that is constantly active and is only partially ornot at all regulated by ligand. Such independence from the normalsignals controlling cellular growth is the essence of tumorigenesis.Tumor types I, II, and III appear to be of the second subset.

METHODS

DNA Hybridization

DNA was purified using Proteinase K-SDS and extracted with phenol andchloroform (Vogelstein et al., 1987). DNA (4 ug) was digested with EcoRI(BRL) using buffers and conditions specified by the manufacturer, andafter electrophoresis through a 1% agarose gel, transferred to nylonmembranes (Bio-Rad) using 0.4N NaOH (Reed et al., 1985).Pre-hybridization and hybridization were done as described previously(Vogelstein et al., 1987). Probes were labelled with d-³²PdCTP using theoligolabelling method (Feinberg and Vogelstein, 1984). Repeatedsequences were removed by the pre-association method of Sealey et al.(Sealey et al., 1985).

Library Construction

The genomic library was constructed from D320MG as described previously(Ruppert et al., 1988). After partial Mbo I digestion, DNA wassize-fractionated through sucrose density gradient ultracentrifugation.The fractions containing 17 to 24 kilobase fragments were cloned intothe Barn HI site of Lambda Fix (Stratagene) after partial fill-in of MboI ends. The ligation product was packaged with lambda phage extracts(Stratagene) and used to infect E. coli C600 cells. DNA from theresulting plaques was lifted with Colony Plaque Screen membranes(Dupont, NEN Research Products) and screened with EGF receptor cDNAprobes (Ullrich et al., 1984; Merlino et al., 1985).

To clone the 1.7 kb rearranged fragment, D245 DNA was digested withEcoRI and size selected by electrophoresis on a 1% agarose gel. DNA waseluted from the gel (Vogelstein et al., 1987), ligated to gt10 arms(Promega), packaged with lambda phage extracts and used to infect C600cells. The library was screened with an EGFR cDNA probe and a clonecontaining the 1.7 kb rearranged fragment was identified. To clone theTEG locus, a total genomic library was first made from D245MG DNA andscreened with the 1.7 kb rearranged EcoRI fragment, resulting in a 15 kbphage clone bridging the rearrangement between TEG and EGF receptorloci. The 4.4 kb fragment from this phage clone was then used to screena genomic library made from D259 (a glial tumor cell line without EGFRamplification or rearrangement) to clone the normal TEG locus. Thefragments from D245, EGFR and TEG participating in the recombinationwere subcloned into pBluescript (Stratagene). For sequencing, nesteddeletions were generated using exonuclease III and mung bean nuclease(Henikof et al., 1984). Plasmids were transformed into HB101 cells(F⁺::Tn5) and single stranded DNA prepared using R408 helper phage(Russel et al., 1986). Sequencing by the di-deoxy method was done usinga modified form of 17 polymerase (USB).

Northern Blotting and cDNA Library Construction

For Northern analysis, poly(A)+RNA was isolated from 10 ug total RNA byselection on oligo-dT cellulose, separated by electrophoresis through a1.5% MOPS/formaldehyde gel and transferred in dilute alkali to nylon(Bio-Rad). For construction of the cDNA library, first strand cDNA wasprepared using MMLV reverse transcriptase (BRL) and random hexamerprimers (Pharmacia). The second strand was synthesized using the methodof Gubler and Hoffman (Gubler et al., 1983). The resulting cDNA wasmethylated with EcoRI methylase and ligated to EcoRI linkers (NewEngland Biolabs). The linked cDNA was cleaved with EcoRi, andfragments >1.0 kb were isolated following electrophoresis through a 1%agarose gel. This cDNA was ligated into ZAP (Stratagene) and packagedwith phage extracts (Stratagene). The library was screened with an 0.7kb TaqI-EcoRI fragment derived from the EGFR end of the 1.7 kb EcoRIfragment of D245 (See FIG. 5A). Plasmids containing inserts were derivedfrom phase plaques by using the excision process recommended by themanufacturer.

Ribonuclease Protection

Total RNA was isolated by the acid-guanidium extraction method describedby Chomcynski and Sacchi (Chomczynski et al., 1987). ³²P-labeled RNAtranscripts were generated in vitro from subclones of EGFR cDNA using T3or T7 RNA polymerase. The probes used were: Probe I: a 910 bp SmaI-ClaIfragment of pEl5 (Merlino et al., 1985); Probe II: a 730 bp EcoRI-Bam HIfragment from-pE7 (Xu et al., 1984); Probe III: a 970 bp Bam HI-EcoRIfragment from pE7. Ribonuclease protection was performed as described(Winter et al., 1985) with the following modifications: hybridizationswere performed in a final volume of 10 ul; only RNase A at 12.5 ug perml was used; and the RNase A and Proteinase K digestions were performedat room temperature for 30 minutes.

PCR amplification of cDNA Products

To generate first strand cDNA, 50 pmol of the 3′ primer was annealed to1 ug of total RNA by cooling from 60° C. to 37° C. over ½ hour in thepresence of 400 uM of each deoxynucleotide, reverse transcriptase buffer(BRL), 1 U of placental RNase inhibitor (Promega) in a total volume of25 ul. This was followed by the addition of 20 U of MMLV reversetranscriptase (13RL) and incubation at 37° C. for 10 min. PCR wascarried out on this sample by diluting the sample to 50 ul, adjustingthe final MgCl₂ concentration to 2.5 mM, and adding 50 pmol of the 5′primer and 2.5 U of Taq polymerase (Cetus). Thermal cycling was done at93° C. for 1 min., 40° for 2 min., and 72° for 2 min. for 35 cycles. Thesample was then subjected to electrophoresis in a 2% agarose gel.Following staining with ethidium bromide, the band was excised, the DNAeluted as described previously (Vogelstein et al., 1987) and subclonedinto pBluescript (Stratagene) for sequencing. At least two clones foreach PCR product were sequenced and some samples were sequenced directlyby the method of Winship (Winship et al., 1989). For tumors D270, D317,and D397, primer set A was used, consisting of 5′-AGTCGGGCTGGAGGA-3′ and5′-ACTGATGGAGGTGCAGT-3′. For tumors D256 and D298, primer set B was usedconsisting of 5′-(C T G) C A G G T C T G C C A T G C C T T G-3′ and5′-(GGT)ACCATCCCAGTGGCGATG-3′. The sequences in parentheses were notpresent in the EGFR sequence and were added to complete either a Pst Ior Kpn I restriction site.

FIG. 4—Deletions in the EGF receptor gene in human gliomas.

FIG. 4A is an EcoRI map of the Human EGF receptor gene. The sizes of thefragments are indicated in kilobases. Representative phage clones usedto assemble the map are shown below it.

FIG. 4B is the deduced EGFR gene structure of five glioma xenografts.The numbers to the left correspond to the glial tumors described in thetext. The solid lines indicate sequences present in the tumors, whilethe approximate points of deletion are indicated by x's.

FIG. 4C represents a Southern blot hybridization with phage clonesdemonstrating deletions in the EGF receptor gene. The blots werehybridized with radio-labelled phage inserts, and the numbers above eachblot refer to the phage clone used as the hybridization probe.Rearranged fragments are indicated with an asterisk (*). The tick marksto the right of each autoradiograph refer to the sizes of markerfragments in kilobases which are given on the extreme right.

The lanes contained DNA according to the following key:

N: DNA from normal cells

A) D245

B) D256

C) D270

D) D320

E) D298

F) D370

FIG. 5—Characterization of the rearranged fragment from D245.

FIG. 5A is an EcoRI restriction map of genomic clones containing the 1.7kb rearranged fragment from D245MG and the corresponding unrearrangedfragments from the EGF receptor gene and TEG locus. The open boxindicates sequences derived from the EGF receptor gene and the shadedbox represents the TEG locus. The numbers refer to the sizes of theEcoRi fragments in kilobases.

FIG. 5B shows a partial nucleotide sequence of the area flanking thesite of recombination. Other features based on this sequence are shownin FIG. 5C. An 18 nucleotide A-T rich motif is repeated four times(rpt1-rpt4) in the rearranged D245 fragment but only once in the TEGfragment; an insertion has been made to optimize alignment. Sevennucleotides are present at the site of recombination (“Break point”)which cannot be aligned with either of the recombination partners.Portions of the Alu repeats present in the TEG and EGF receptor gene areunderlined; the arrowhead indicates the orientation of these repeatsaccording to Deininger et al. (Deininger et al., 1981). Numbers to theright are relative nucleotide positions with respect to the D245sequence.

FIG. 5C schematically shows the presence of Alu repeats (arrows), theA-T rich motif (open box labeled Al) and exon from the EGF receptor(filled box corresponding to EGFR nt 1818-1908) in the 1.77 kb EcoRIrearranged fragment from D245 and the corresponding unrearrangedfragment from the EGF receptor gene and TEG locus.

FIG. 5D illustrates chromosomal localization of the TEG locus. Ahuman-mouse somatic cell hybrid panel containing various deletions ofhuman chromosome 7 (20-21) was digested with EcoRI and blotted to anylon membrane. It was first hybridized with the 2.2 kb EcoRI fragmentfrom the TEG locus (top) and then hybridized with a 1.9 kb EcoRI genomicfragment from the EGFR locus (bottom).

The somatic cell hybrid clones used (and the regions deleted within thehybrids) were:

Lane 1: 5387-3 cl 10 (intact chromosome 7);

Lane 2: It A9 2-21-14 (7pl4-pter);

Lane 3: RuRag 6-19 (7 cen-pter);

Lane 4: 2068 Rag 22-2 (7 q22-pter);

Lane 5: 1059 Rag 5 (7 q22-q32);

Lane 6: 7851 Rag 10-1 (7q22-q32);

Lane 7: 194 Rag 6-13-3(7 q32-qter);

Lane 8: Rag (mouse DNA only);

Lane 9: 4 ug of normal human DNA.

FIG. 5E describes a possible mechanism for intrastrand recombination inD245 involving oppositely paired Alu repeats based on the model proposedby Lehrman et al. (Lehrrnan et al., 1985). Features of the TEG and EGFreceptor fragments from FIG. 5C are reproduced here. The oppositelyoriented Alu repeats in the EGF receptor gene and TEG locus onchromosome 7p could base pair forming a stem loop structure.Recombination at the stem would result in the rerangement seen in D245.

FIG. 6—Characterization of the transcript in D245.

FIG. 6 is a Northern blot hybridization demonstrating abnormaltranscript sizes in D245. Two and one-half ug of poly A+ selected RNAfrom placenta and tumor D245 were electrophoresed in a 1.5% formaldehydegel and transferred to nylon membranes. The blot was hybridized with aprobe specific for the intracytoplasmic domain of the EGF receptor. Thenumbers to the right and left indicate the size of the bands in kb. Themajor band in the D245 sample is 4.8 kb; there is also a faint band ofunclear derivation at 5.5 kb which was not derived from the normal EGFRmRNA, since it did not hybridize to an extracytoplasmic domain probe,

Therefore a separate blot that was hybridized with a probe from theextracytoplasmic domain (the 730 bp EcoRi-BamHI fragment of pE7.

FIG. 7—Characterization of transcripts by RNase protection.

FIG. 7A is a schematic drawing of the probes used. The 5.5 kb EGFreceptor cDNA is diagrammed above. The striped box is the signalpeptide, followed by the extracytoplasic domain which is divided intofour domains (1-4), with the cysteine rich regions as indicated. Thesolid box is the transmembrarne domain followed by the intracytoplasmicdomain containing the kinase domain. The thin lines are the untranslatedregions.

FIG. 7B presents the deduced transcript structures from the RNaseprotection experiments. Symbols are as in FIG. 4B.

FIG. 7C depicts RNase protection experiments using RNA from the glialtumor xenografts. The numerals I-III refer to the probes in FIG. 7A. Thenumbers to the right refer to the sizes of the protected fragments (seetext).

The lanes contain RNA from: A) D245; B) D256; C) 2709; D) D320; E)D298;F) D317; tRNA (negative control); and H) undigested probe.

FIG. 8—PCR analysis of the gene products from the altered region.

FIG. 8A shows gel electrophoresis of polymerase chain reaction products.The gel shows {fraction (1/10)} of the reaction after 35 cycles.

The lanes contain products from: A) 270; B) 317; C) 397; D) 256; E) 298;F). Primer set A was used for D270, D317, and D397; primer set B wasused for D256, D298, and D320 (see Experimental Procedures). The numberrefer to the sizes of fragments in base pairs judged fromco-electrophoresed markers.

FIG. 8B shows a sequence of the PCR products from tumors D270, D317, andD397.

FIG. 8C illustrates the sequence of the PCR products from D256 and D298.The numbers and asterisks refer to the EGF receptor cDNA nucleotidesflanking the deleted area. The sequence to the right and left of thoseshown in the figure were the same as in normal EGFR cDNA.

H. Antibody Production

1. Mice

Mice for the production of hybridomas (later to be fused into a myeloma)were injected according to the schedule below. Mice populations were atleast 5 and generally up to 10 in each immunization arm with both Type Iand Type II EGFR deletion mutants.

Day Immunization Arm I:  0 100 ug pep − 200 ug KLH + CFA id 15 30 ug pep− 60 ug KLH + IFA ip 30 10 ug pep − 20 ug KLH + IFA ip 44 2.5 ug pep iv105  1 ug iv 138  1 ug iv 166  1 ug iv prefusion titer: greater than1/2000-1/4000 181  1 ug iv 186  1 ug iv 187  1 ug iv 191  fusion Arm II: 0 50 ug pep − 100 ug KLH + CFA id 14 15 ug pep − 30 ug KLH ip 28 1 ugpep iv titers: alpha peptide Type I: gtr. than 1/8,000 alpha peptideType II: gtr. than 1/8,000 62 1 ug pep iv 69 1 ug pep iv 70 1 ug pep iv72 fusion; prefusion serum samples available Arm III:  0 50 ug pep − 100ug KLH + CFA id 21 1 ug pep iv prefusion titer: alpha peptide Type I:gtr. than 1/2,000-1/5000 alpha peptide Type II: gtr. than1/4,000-1/19,000 40 1 ug pep iv 47 1 ug pep iv 48 1 ug pep iv 50 fusionArm IV:  1 50 ug pep − 2 − KLH + CFA id 18 100 ug pep − 2 − KLH + IFA id32 50 ug pep − 2 − KLH + PBS ip 39 bled and titered 46 50 ug pep − 2 −KLH in PBS ip 49 fusion Arm V:  1 500 ug pep − 2 − KLH + CFA id 18 100ug pep − 2 − KLH + IFA id 32 50 ug pep − 2 − KLH + PBS ip 39 bled andtitered 50 2.5 ug free pep − 2 iv 54 fusion Abbreviations: KLH = keyholelimpit hemocyanin CFA = complete Freund's adjuvant IPA = incompleteFreund's adjuvant id = intradermal ip = intraperitoneal pep = peptidetype I or type II PBS = phosphate buffered saline

Hybridoma supernatants were screened using solid phase radioimmunassyusing 0.5 ug/well of pep-2. Hyperimmune sera, specific for pep-2 versuspep-1 preimmune sera was used as baseline controls.

Over twenty-five murine hybridomas making monoclonal antibodies to theEGF receptor mutant peptide (class A) have been isolated.

2. Rabbits

Immunization Schedules

Rabbits were injected with synthetic peptides of the deletion mutantcoupled to keyhole limpet hemocyanin initially, and after two months,three further injections at one month intervals. The immunizationpattern was performed with rabbits according to the following schedule:

Day −7 Pre-immune bleed (50 ml per rabbit via ear vein) Day 0 250 ugpeptide-KLH conjugate of 1 ml in phosphate-buffered saline; completeFreund's adjuvant (1:1) per rabbit injected subcutaneously with 200lamdba at 5 different sites Day 30 50 ug peptide-KLH conjugate in 1 mlof phosphate -buffered saline; incomplete Freund's adjuvant (1:1) perrabbit injected subcutaneously with 200 lambda at 5 different sites Day39 Rabbits bled, serum harvested, and stored in 1 ml aliquots at −135°C.

In another experiment, a slightly different schedule was followed.

Three New Zealand white rabbits were immunized with peptide conjugatedto KLH. On Day 0, subcutaneous immunization was administered with 250 mgpeptide conjugated to KLH in 1 ml of a 1:1 emulsification ofphosphate-buffered saline (PBS) and complete Freund's adjuvant. Eachrabbit was injected with this dose at 4 separate sites. On Day 33, eachrabbit was boosted at 4 separate sites with 50 mg peptide conjugated toKLH in 1 ml of a 1:1 emulsification of PBS and incomplete Freund'sadjuvant. Antisera were obtained by bleeds on Days 40 and 43.

I. Titration of Antibodies

Antibody titers against peptide were determined in ELISA. Briefly, aconcentrated solution of peptide (1 mg/1 ml in PBS) was diluted to 10mg/ml in 200 mM NaHCO₃ buffer (ph 9.2), and 50 ml were added to 96platewells of polyvinyl chloride (Dynatech Laboratories, Inc., Chantilly,Va.). The peptide solution was incubated in the plate wells overnight at4° C. The peptide solution was discarded, and the plates were washed 3times with Hanks' buffered salt solution containing 0.05% Tween 20(Sigma, St. Louis, Mo.). Nonspecific binding was then blocked for 1 hrat room temperature with 200 ml of 0.5% bovine serum albumin in PBS. Theplates were washed as above. Pre-immune rabbit sera or antisera (100 mlof dilutions in Hanks' buffered salt solution with 0.5% BSA) were addedfor 2 hr at room temperature. The plates were washed and 50 ml ofperoxidase-conjugated goat antirabbit IgG (Zymed Laboratories, Inc., SanFrancisco, Calif.) was added for 1 hr at 37° C. The plates were washedand 100 ml of substrate solution was added. The substrate solution wasprepared by adding 15 mg of o-phenylenediamine (Sigma) to 1 ml methanol,with subsequent addition of 49 ml of deionized water and 50 ml ofhydrogen peroxide. Incubation was at room temperature for 15-20 min.,and the well absorbances were then measured in an automated plate reader(Titertek Multiskan MCC/340, Flow Laboratories, McLean, Va.) at 492 nm.The ELISA could also be performed with purified antipeptide antibody.The half-maximal titer in this assay was 0.5 mg antipeptide IgG/ml ofPBS.

All three rabbits immunized with peptide-KLH conjugate exhibited amarked IgG response as assessed by ELISA against uncoupled peptide boundto plates (FIG. 9). The half-maximal titers (the antiserum dilution atone-half maximal absorbance) varied from 1:6000 to 1:50,000. The bindingreaction was specific, as the pre-immune serum from each rabbit wasnonreactive; a nonreactive baseline was also obtained when the antiserawere reacted with a second 14-amino-acid pep tide of different sequence(Pep-1; sequence:H-Asn-Leu-Leu-Glu-Gly-Cys-Thr-Gly-Pro-Gly-Leu-Glu-Gly-Cys-OH).

J. Purification and Characterization of Antipeptide Antibody

The antipeptide antibody was purified from antiserum by apeptide-Sepharose affinity column with elution by acidic pH. Theaffinity column was prepared by coupling 5 mg of peptide to cyanogenbromide-activated Sepharose (Sigma), as described in the Pharmacia(Piscataway, N.J.) protocol. The extent of coupling was 100%, asdetermined by BCA protein assay (Pierce, Rockford, Ill.) of the solutionoverlying the gel. Ten millititers of antiserum from rabbit 396 (therabbit with the highest half-maximal titer of 1:50,000 in ELISA inreaction against peptide) were passed over the column, and the columnwas washed extensively with 500 ml of PBS. Elution was with 100 mMglycine buffer (pH 2.5) with immediate neutralization into 0.4 M Hepesbuffer (pH 7.4). A yield of 6 mg of pure IgG was obtained from astarting load of 10 ml of rabbit antiserum 396 (the antiserum with the1:50,000 half-maximal titer). Lower yields were obtained withpurification of antipeptide IgG from antisera of lower half-maximaltiters. Elution of active antibody could also be accomplished with 3.5 MMgCl₂ (pH 3.5 or pH 6.5). The activity of the antibody eluted under thedifferent conditions was similar, as assessed by ELISA against freepeptide.

Control IgG was purified from rabbit pre-immune sera by proteinA-Sepharose affinity chromatography. The size and homogeneity of thepurified antibodies were monitored under nondenaturing conditions bysize exclusion HPLC and under denaturing condition by SDS-PAGE. HPLC wasperformed on a calibrated 1×30 cm Waters 300 SW column (previouslycalibrated with standards in the size range of M_(r) of 20,000-400,000daltons). Protein elution was followed at 215 nm. SDS-PAGE utilized a10% resolving gel in the SDS-discontinuous buffer system of Laemmli(Laemmli, Nature (1970), 270:680-688). The protein bands were visualizedwith Coomassie blue staining. The eluted antibody was identified as IgGby apparent molecular weights on a size exclusion HPLC column and SDSgel (data not shown). Purity was greater than 98%.

K. Immunocytochemical Detection of Mutant EGFR Expression

The affinity-purified antibody was characterized in immunocytochemistryusing frozen tissue sections and the avidin-biotin complex method, asdescribed (Humphrey, et al., Cancer Res. (1988), 48:2231-2238). Thetissues tested included a range of normal fetal and adult tissues,carcinomas (prostatic, bladder, breast, and lung), glioma biopsies(-Bx), and gliomas grown in xenograft form in nude mice (-X). The normaltissues, carcinomas, and glioma biopsies were from the Duke UniversityMedical Center Tissue Bank. The glioma xenografts were grown asdescribed (Humphrey, et al., Cancer Res. (1988), 48:2231-2238). Theaffinity purified antipeptide antibody and the purified pre-immunerabbit control IgG were used at a concentration of 1-2 mg/ml, asdetermined by initial titration experiments. The antibody could be usedin antiserum form with an optimal titer in immunocytochemistry of1:3000. n(ab′)₂ fragments of normal rabbit IgG and the antipeptideantibody were also tested in immunocytochemistry on glioma D-270 MG-Xand skin. F(ab′)₂ fragments were generated and purified as described(Colapinto, et al., Cancer Res. (1988), 48:5701-5707). Also used inimmunocytochemistry of the glioma biopsies was rabbit antipeptideantibody reactive with the intact EGFR (Product No: OA-11-852, CambridgeResearch Biochemicals, Valley Stream, N.Y.). This antiserum was used ata dilution of 1:1000, as established by initial titration experiments.The antipeptide IgG reacted with the native mutant EGFR in frozen tissuesections fixed briefly with acetone. The antibody specificallyrecognized only the mutant glioma EGFR and not the intact A431-Xsquamous cell carcinoma EGFR in both xenograft (data not shown) andbiopsy tissue (Table 1). This immunostaining of the mutant EGFR wasspecific, as purified pre-immune IgG was nonreactive, and preincubationof antipeptide IgG with excess peptide blocked with immunocytochemicalstaining. In both glioma biopsy and xenograft tissues which bound theantipeptide antibody, the immunostaining was localized to the cytoplasmand cell surface; virtually every tumor cell exhibited immunoreactivity.

Screening of normal and neoplastic tissues by immunocytochemistry withthe antipeptide antibody revealed positivity only in a subset ofglioblastomas (Table 1). Immunoreactive mutant EGFR was identified intwo human glioma biopsies (D-270 MG-Bx and D-317 MG-Bx) and thecorresponding xenografts (D-270 MG-X and D-317 MG-X) known to amplifythe mutant EGFR gene and express mutant EGFR protein. Four additionalglioblastoma biopsies exhibited immunostaining with the antipeptideantibody; one of these (D-397 MG-Bx) was subsequently tested using anRNA-based PCR assay and shown by sequencing to possess the same deletionmutation as gliomas D-270 MG-Bx and D-317 MG-Bx.

Further evidence for the selectivity of this antibody was obtained inthis screen of glioma biopsies, as 27 of 27 biopsies reactedspecifically with rabbit antipeptide antibody against the intact EGFR;but only those gliomas with the proven (3 cases) or suspected (3 cases)deletion mutation reacted with the antifusion junction peptide antibody.A range of fetal and adult normal tissues and carcinomas failed to reactwith the antipeptide antibody, as assessed by this method.

TABLE 1 Antipeptide antibody immunostaining of normal and Tumor tissuefrozen sections Normal human Human neoplastic tissues Reactivity*tissues (biopsies) Reactivity Adult Brain 0/2 Glioblastoma  6/35 Muscle0/1 Lung Carcinoma 0/2 Skin  0/3⁺ Prostatic Carcinoma 0/2 Kidney 0/3Breast Carcinoma 0/2 Spleen 0/2 Bladder Carcinoma 0/2 Placenta  0/11Fetal Brain 0/1 Kidney 0/1 Liver 0/1 *Results are expressed as number ofpositive cases/total number of cases examined. ⁺Weak Fc receptor bindingwas observed in one case in epidermal epithelium. This immunostainingwas abolished with the use of F(ab′)² fragments. Note: Lack ofreactivity with normal adult brain and skin was confirmed byimmunoprecipitation.

L. Binding of ¹²⁵I-Labeled Antipeptide IgG to Tumor Membranes ExpressingIntact and Mutant EGFR

The purified antipeptide antibody and pre-immune IgG were both labeledwith ¹²⁵I at a specific activity of about 1.6 mCi/mg using a variationoff the iodogen method as described previously (Colapinto, et al.,Cancer Res. (1988), 48:5701-5707). Radioiodinated proteins (22 ng, 50-70k cpm) were incubated for 2 hr at 4° C. in triplicate with 200 ml of 20mM Hepes (pH 7.4) containing 0.1% BSA and 40 ml of a 1-mg/ml suspensionof microsomal membranes. The membranes tested were from D-270 MG-X, aglioma tumor expressing the in-frame deletion-mutant EGFR; A431-X, asquamous cell carcinoma overexpressing the intact EGFR; and D-245 MG-X,a tumor containing or expressing an EGFR molecule which lacks most ofthe extracellular domain and serves as a negative tissue control.Following the incubation period, the membranes were separated fromunbound activity using 0.22-mm cellulose acetate centrifuge filter units(Spin-x, Costar, Cambridge, Mass.) washed twice with 1 ml of theincubation buffer. The filters had been pretreated by a 30-minincubation at room temperature followed by 3 washes with buffer. Usingthis procedure, nonspecific binding of radioactivity to the filters was<0.2%. Filters and washes were assayed for ¹²⁵I activity in similarcounting geometries using an automated gamma counter.

Direct binding of radioiodinated antipeptide IgG to membranes expressingthe D-270 MG-X mutant EGFR was significantly higher than to membranesexpressing intact EGFR (in A431-X) or a v-erb B-like EGFR (in D-245MG-X) (Table 2). The specificity of the antipeptide binding reaction wasdemonstrated by assessing nonspecific binding using ¹251-labeledpurified pre-immune IgG. The selectivity of the antipeptide antibody ishighlighted by comparing the percentage specific binding values; thelevel of antipeptide antibody binding to the mutant EGFR in the D-270MG-X membranes is markedly higher than the low reactivity with intactEGFR, which is similar to that of the negative tumor membrane control.

TABLE 2 Antipeptide antibody binding to membranes of tumors Expressingthe deletion mutant EGFR, intact EGFR and v-erb B-like EGFR EGFR PercentPercent specific IgG Tumor Structure Binding Binding Antipeptide 270In-frame deletion 30.2 28.2 mutant Pre-immune 270 In-frame deletion 2.0mutant Antipeptide A431 Intact 8.5 4.9 Pre-immune A431 Intact 3.6Antipeptide 245 v-erbB-like 6.8 3.8 Pre-immune 245 v-erbB-like 3.0Percent binding represents mean fraction of input counts (of atriplicate assay) associated with the membranes. Percent specificbinding represents binding of ¹²⁵I-labeled antipeptide IgG minusnonspecific binding determined from association of preimmune IgG withmembranes. Glioma D-270 MG-X membranes express the in-framedeletion-mutant EGFR and squamous cell carcinoma A431-X membranesoverexpress the intact EGFR; glioma D-245 MG-X membranes express#v-erbB-like EGFR which lacks most of the extracellular domain andserves as a negative tissue control (Humphrey, et al., Cancer Res.(1988), 48:2231-2238; Wong, et al., J. Cell Biochem., Suppl. 13B, Abst.149).

M. Reaction of Antipeptide IpG with Mutant EGFR but not Intact EGFR

The molecular specificity of the antipeptide IgG was tested byimmunoprecipitation reaction. The binding in this reaction was followedby EGFR autophosphorylation, SDS-PAGE, and autoradiography, essentiallyas described. Monoclonal antibody 528 (Ab-1, Oncogene Science, Inc.,Manhasset, N.Y.) was used as a positive control, as it willimmunoprecipitate both intact and mutant EGFR. Briefly, Triton X-100detergent solubilized intact A431-X EGFR and mutant D-256 MG-X and D-270MG-X EGFRs were immunoprecipitated with monoclonal antibody 528 orpurified antipeptide antibody. Autophosphorylation with ³²P-ATP wasfollowed by SDS-PAGE with a 7.5% resolving gel. The gel was fixed anddried under vacuum and exposed to X-ray film at −70° C. The antipeptideantibody was used at 20 mg purified IgG or at 100 ml antiserum inbinding to Protein A-Sepharose for the immunoprecipitation reaction.

The antipeptide antibody reacted with mutant EGFR in adetergent-solubilized state, as judged by immunoprecipitation, withautophosphorylation and SDS-PAGE (FIG. 10). The positive controlimmunoprecipitation was with monoclonal antibody 528, which is directedagainst the EGFR external domain. This antibody reacted with both theintact A431-X and mutant D-270 MG-X EGFR. The antipeptide antibody, incontrast, specifically immunoprecipitated the 145-kDa mutant EGFR inglioma D-270 MG-X and failed to immunoprecipitate the intact A43 1-XEGFR. Purified preimmune IgG did not immunoprecipitate the mutant EGFR.

N. Effect of Antipeptide IgG on ¹²⁵I-Labeled EGF Binding To the MutantEGFR and on Mutant EGFR Kinase Activity

The effect of the purified antibody on ¹²⁵I-labeled epidermal growthfactor (EGF) (New England Nuclear, Doraville, Ga.) binding wasdetermined using the reaction mixture of microsomal membranes and¹²⁵I-labeled EGF, as described (Humphrey, et al., Cancer Res. (1988),48:2231-2238). To determine the effect of antibody on EGFR kinase, thephosphorylation of EGFR in D-270 MG-X and A431-X membranes was performedas described by Davis and Czech (Davis, et al., J. Biol. Chem. (1985),260:2543-2551). Briefly, the phosphorylation was carried out in a volumeof 50 ml consisting of membranes (5-10 mg), 20-mM Hepes (pH 7.4), 4 mMMnCl₂, 10 mM MgCl₂, 1% DMSO, ±400 ng of EGF, ±normal rabbit IgG,±antipeptide antibody. The mixture was incubated for 20 min at roomtemperature and then cooled to 4° C. [q-³²P]-ATP (1.5 mM; 10-15,000cpm/pmol) was added and the reaction was stopped after 3 min by adding25 ml of sample buffer containing sodium dodecyl sulfate andbetamercaptoethanol. The samples were subjected to electrophoresis on7.5% polyacrylamide gels (Laemmli, Nature (1970), 270:680-688), and theextent of receptor phosphorylation was visualized by autoradiography.

The anti-synthetic peptide antibody did not affect binding of¹²⁵I-labeled EGF to the mutant EGFR, nor did it affect either basal orEGF-stimulated intrinsic kinase activity. Specific binding of¹²⁵I-labeled EGF, defined as counts of total ¹²⁵I EGF binding minuscounts not displaced by a 100-fold excess of unlabeled EGF, did notchange with addition of antibody (data not shown). This may be due tothe location of the fusion junction at a site distant from the EGFbinding domain at residues 351-364 (Wu, et al., J. Biol. Chem. (1989),264:17469-17475). The kinase activity of the mutant EGFR did not changewith the presence of antibody, as assessed by band intensity inautoradiography of an SDS-polyacrylamide gel (data not shown).

O. Internalization of Antipeptide IgG

Binding of antipeptide IgG to live tumor cells was determined byimmunofluorescence assay. D-270 MG-X or A431-X cells were prepared bydissociation from tumor xenografts grown subcutaneously in athymic mice.Briefly, subcutaneous tumors were removed from the mouse and the viableportions were separated from areas of necrosis. The viable tissue wasminced with scissors and dissociated into a single cell suspension byincubating 1 hr in 0.8% collagenase at 37° C. in a trypsinization flask.The cells were washed twice with cold Richter's zinc-option mediumcontaining 1% normal goat serum. One million viable cells, as assessedby trypan blue, were incubated 30 min at 4° C. with zinc-option mediumplus 10% normal goat serum to block nonspecific binding. The cells wereincubated 30 min at 4° C. with 100 ml of antipeptide antibody (20 mg perml) or pre-immune IgG. After washing 3 times with zinc-option mediumplus 1% normal goat serum, the cells were incubated with 100 ml offluorescein-labeled goat antirabbit IgG for 30 min at 4° C. The cellswere washed as above and resuspended in 500 ml of cold zinc-optionmedium plus 1% normal goat serum. One-hundred-microliter aliquots wereremoved and warmed to 37° C. for 10 to 60 min and observed with a Zeissfluorescent microscope.

The anti-synthetic peptide antibody rapidly bound the surface mutantEGFR expressed on live glioma D-270 MG-X cells, and this binding wasreflected by a rimming pattern of the immunofluorescent secondaryantibody label. Internationalization rapidly ensued and was manifestedas a speckled intracytoplasmic morphology. Internalization with loss ofthe peripheral plasma membrane rimming was complete by 60 min at 37° C.

P. Immunoprecipitation of EGFR

EGFR from xenograft tissue was solubilized and immunoprecipitated asfollows. Ten mg of frozen (−70° C.) xenograft tissue were homogenized in1 ml of ice-cold solubilization buffer composed of 20 mM iodoacetate,and Aprotmin at 1 mg/ml. After 2 h at 4° C., the preparation wascentrifuged for 15 min at 4° C. in a Beckman tabletop centrifuge at12,000×g. The supernatant was used in EGFR immunoprecipitation.Immunoprecipitation was performed with monoclonal antibody Ab-1 (clone528: Oncogene Science, Inc.), reactive against the normal externaldomain of EGFR.

Monoclonal antibody Ab-1 (528) is an IgG2a which inhibits EGF binding toits receptor. For each reaction mixture, 5 ug of monoclonal antibodyAb-1 (528) or 15 ul of undiluted antiserum containing polyclonalantibody were bound to 2 mg of Protein A-Sepharose 4B by incubation in115 mM sodium phosphate buffer, pH 7.4, for 30 min at room temperature.Antibody-Protein A-Sepharose complex was washed 3 times with 115 mMsodium phosphate buffer, pH 7.4 EGFR immunoprecipitation was performedwith 500-ul aliquots of solubilized xenograft tissue, 500 ul of 115 mMsodium phosphate buffer, pH 7.4, and the antibody-Protein A-Sepharosepellet.

After pellet resuspension, overnight incubation was done at 4° C.Immunoprecipitates were-washed 3 times with 1 ml of solubilizationbuffer, and the pellet was used for autophosphorylation.

Q. Autophosphorylation of EGFR

Autophorylation of the immunoprecipitated EGFR was performed. TheEGFR-antibody-Protein A-Sepharose pellets were incubated with 30 ul ofsolubilization buffer plus 2 mM MnCl₂ and 3 uCi of gamma-[³²P]ATP (NewEngland Nuclear; 2000 to 3000 Ci/mmol). After reaction for 10 min at 4°C. on ice, the reaction was terminated by the addition of 30 ul of 2×Laemmli SDS-PAGE sample buffer with 2% B-mercaptoethanol. Samples wereboiled for 3 min and centrifuged. Supernatants were used in loadingSDS-polyacrylamide gels.

R. Immunoblot Analysis

Western immunoblot analysis was performed with the use of a semidryhorizontal electrophoretic transfer system with graphite electrodes (LKBMultiphor II Nova Blot System). Briefly, frozen A431 and gliomaxenografts were solubilized in SDS-gel sample buffer, boiled, andelectrophoresed in a 7.5% SDS-polyacrylamide gel. The proteins weretransferred to nitrocellulosse at 150 mA for 2 h, andimmunohistochemical detection of EGFR was accomplished with antibody 528and polyclonal antisera from rabbit no. 4 directed against a type IIpeptide.

Polyclonal antibodies against the Type II mutants are tissue specificwithin the limits of present testing. See, FIGS. 9 and 10.

S. Immunohistochemical Analysis of Normal and Neoplastic Human Tissue

Purified Mabs were screened against acetone fixed HC, 3T3, and A431monolayers, or acetone-fixed frozen sections of D256 MG and D245 MGhuman glioma xenografts passaged in athymic rats. It was determined thatMabs L8A4 and Y10 were the most consistent and optimal reagents forimmunohistochemistry, and were incorporated into an antibody panelconsisting of Pep 3-affinity purified rabbit antiserum, Mab 528, and Mab3B4 (pan human tissue positive control); normal IgG or isotype controlsincluded normal rabbit IgG, and murine IgG1 and IgG2a at correlativeconcentrations. As shown in Table 3, tissues examined included 6 casesof prostatic carcinoma, 11 cases of breast carcinoma, and 31 cases ofglioma. The tumor types studied were chosen on the basis of reportedEGFR gene amplification incidence (Humphrey et al, 1988, Merlino et al,1985) and to complement our previous analysis of non-small cell lungcarcinomas with Pep 3 affinity-purified polyvalent rabbit anti Pep 3antiserum (Garcia de Palazzo et al, Cancer Research, 53:3217-3220,1993). As shown in Table 3, 0/6 prostatic carcinomas expressed variantType II mutant of EGFR as detected by anti Type II mutant of EGFR seraor Mabs, whereas 3/6 expressed wild type EGFR as detected by Mab 528.Among the panel of 11 breast tumors, 3 cases (two infiltrating ductalcarcinoma, one ductal carcinoma) were found to exhibit a primarilycytoplasmic staining pattern, with focal areas of membrane positivitywith Mab 528 and the anti Type II mutant of EGFR Mabs as opposed toserial sections stained with irrelevant control IgG1. To furtherinvestigate the expression of EGFRvIII by breast carcinomas, we isolatedmRNA from sections of 10/11 of the same tissue blocks studiedimmunohistochemically, and analyzed for expression of EGFRvIII usingRT-PCR. Primers were selected to amplify a band of 236 bp if theEGFRvIII deletion was present, and with a band of 1037 bp for normalEGFR. Products corresponding to PCR amplification of EGFRvIII mRNA werepresent in 3 of 3 breast carcinoma tissues which were reactive with L8A4Mab immunohistochemically confirming the specificity of Mab L8A4. Inaddition, bands corresponding to EGFRvIII were detected in 5 additionalbreast carcinomas which had demonstrated no immunohistochemicalreactivity with Mab L8A4. Therefore, the presence of EGFRvIII mRNA wasdetected by RT-PCR in 8 of 10 of the 11 beast carcinoma tissuesevaluated by immunohistochemistry. Two of the 8 tissues containing TypeII mutant of EGFR co-expressed normal EGFR. Our increased detection ofEGFRvIII by RT-PCR is compared to immunohistochemical analysis reflectsthe well-established greater sensitivity of PCR-based assays.

TABLE 2 Reactivity of frozen human tumor and normal tissue sections withanti-EGFR and Type II Mutant of EGFR Mabs Positive Reactivity with:Anti-EGFR Type II Mutant of EGFR Tumor or Normal Tissue number of Mab528 Mabs L8A4 & Y10^(a) Classification cases +/Σ % +/Σ % CommentProstatic carcinoma 6 3/6 50 0/6 0 Breast carcinoma 11  3/11 27  3/11 27primarily cytoplasmic infiltrating ductal 10  2/10 20  2/10 20 stainingintraductal 1 1/1 1/1 Glioma 31 24/31 77 16/31 52 membranous andcytoplasmic anaplastic astrocytoma 7 4/7 57 1/7 14 staining withperivascular gliosarcoma 3 2/3 66 2/3 66 accumulation (17/31, Mabglioblastoma multiforme 21 18/21 86 13/21 62 528; 5/31, Mabs L8A4 andY10) Normal Tissues colon, kidney, testes, 3 0/3  0/3 nonspecific Kupfercell lung, cerebellum, uptake cerebral cortex, liver ovary, skin,peripheral 2 0/2^(b) 0/2 nerve, bone marrow, lymph node spleen 4 4/4^(c)4/4^(c) see footnote c ^(a)Mabs L8A4 and Y10 reacted identically with10/21 glioblastomas and 2/3 gliosarcomas. Mab L8A4 positively stained anadditional 3 glioblastomas and 1 anaplastic astrocytoma; in these lattercases Y10 reactivity was marginal and interpreted as negative. ^(b)Mab528 (anti-normal EGFR) reacted with endothelial cells and macrophage inboth peripheral nerve samples. ^(c)Immunohistochemically, all fourspleens exhibited light, diffuse staining in B cell areas aroundgerminal centers and in the red pulp with Mabs 528, L8A4, and Y10, whichwas more pronounced than in primary isotype control section. Extensiveanalysis of two of these spleen samples by lysate preparation, SDS-PAGEand Western blot, and Facs # analysis of LSM gradient preparedlymphocytes, and of one of these two samples by RT-PCR failed to yieldany evidence of either normal or Type II Mutant of EGFR protein or RNAexpression.

Within the panel of gliomas examined (Table 3), normal epidermal growthfactor receptor was identified by Mab 528 in 4/7 anaplastic astrocytomasand 18/21 glioblastomas (including 2/2 gliosarcomas). In contrast, typeII mutant of EGFR was immunolocalized in 1/4 anaplastic astrocytomas and15/21 glioblastomas, and 2/3 gliosarcomas. Cytologic localization ofboth normal (in those tumors positive with Mab 528 and negative withanti-type II mutant of EGFR Mabs) and mutant proteins indicated apredominance of cytoplasmic staining, although significant variationbetween cytoplasmic and membranous localization was found within thesame tumor. In determining immunopositivity for calculating a labelingindex either cytoplasmic or membranous immunoreactivity was consideredpositive. The labeling index among these tumors by Mab is shown in Table3. These results indicate that only a small proportion (3/21) of gliomaslack normal EGFR expression, and that a subset of EGFR positive gliomas(13/21) express, type II mutant of EGFR as well. Although nuclearimmunoreactivity was commonly found with the Mab Y10, no otherimmunoreagent produced this localization pattern and it was ingored indetermining the Y10 labeling index. An interesting histologic patternidentified in these studies was perivascular accentuation, primarily ofEGFR, but occasionally of type II mutant of EGFR, localization. Thepattern was always fibrillar, and presumably glial in origin, incontrast to the extracellular matrix localization found with antibodiesto proteins such as tenascin. This pattern was found with Mab 528 in 3/7anaplastic astrocytomas, 12/21 glioblastomas, and 2/3 gliosarcomas. Theincidence was lower with anti-type II mutant of EGFR Mabs, being seen in0/7 anaplastic astrocytomas, 3/21 glioblastomas, and 2/3 gliosarcomaswith anti-type II mutant of EGFR Mabs L8A4 and Y10.

METHODS

Immunogens, Immunization Protocols, and Fusions

Production and sequence purity of the synthetic peptides used has beenpreviously published (Humphrey et al, 1990, 1991). For Pep 3, a 14 aminoacid peptide corresponding to the predicted amino acid sequence at thefusion junction,n (LEEKKGNYVVC) was synthesized, purified, and coupledto KLH by AnaSpec, Inc. (San Jose, Calif.). The peptide was coupled byminimally intrusive coupling chemistry via a thiol linkage with theadded terminal cysteine using the heterobifunctional cross-linkerN-succinimidyl bromoacetate, through amide bonds with the carrier(Bematowicz et at, 1986). A 10 amino acid peptide of unrelated structure(CNLLEGCTGP), Pep 1, served as negative control. Structuralcharacterization and purity were determined by amino acid analysis andmass spectroscopy at AnaSpec, Inc., and the Macromolecular StructureLaboratory of the Duke Comprehensive Cancer Center.

Combination immunization protocols, as detailed below, utilized thefillowing immunogens: 1) Pep 3 conjugated to KLH in a 1:1 emulsion ofDulbecco's Phosphate Buffered Saline (DPBS) in Complete Freund'sAdjuvant (CFA; Difco, Detroit, Mich.), Incomplete Freund's Adjuvant(IFA), or in DPBS alone; 2) collagenase-disaggregated D270 MG xenograftcells; 3) 0.02% EDTA-DPBS harvested cultured HC cells; and 4) microsomalmembrane preparations of HC xenograft cells.

Four different immunization protocols ultimately led to the isolation ofspecific, type II mutant of EGFR Mabs. BALB/c female mice (Charles RiverBreeding Laboratories, Stoneridge, N.Y.), 8-15 weeks old at theinitiation of immunization, were used. Protocol 1, (Mabs J2B9, J3F6):day 1, 100 μg Pep 3-KLH-CFA intradermally (id), 4 sites; days 56 and132, 1×10 D270 MG xenograft cells, intraperitoneally (ip); day 157, 30μg Pep 3-KLH, ip, day 161, fusion. Protocol 2, (Mab L8A4): days 1, 25,5×10⁶ HC cells ip; day 40, 30 μg Pep 3-KLH-CFA, id; days 74, 87, 1×10⁷HC cells ip; day 103, 30 μg Pep 3-KLH, ip; day 107, fusion. Protocol 3,(Mab Y10): day 1, 5×10⁶ HC cells ip; day 161, HC microsomal membranesequivalent to 5×10⁶ HC cells (HC membranes 1×) in CFA, subcutaneously(sc); day 175, HC membranes 1× in IFA, sc; day 199, 30 μg Pep 3-KLH-IFAsc; day 213, 5×10⁶ HC cells ip; day 216, fusion. Protocol 4, (Mabs H10,H11), Mab H11, HC membranes 1×, Mab H10, HC membranes 25×): day 1, HC 1×or 25× in CFA, (sc); day 68, 100 μg Pep 3-KLH-IFA+1 mg S. minnesota; day83, HC membranes 2× in 115 mM phosphate buffer, ip; day 177, as for day83+100 μg Pep 3-KLH, ip; day 194, as for day 83+50 μg Pep 3-KLH ip; day197, fusion. All immunized animals were bled at various intervals todetermine titers; in general, reciprocal 50% endpoint titers in excessof 5000 vs Pep 3 and the receptor target were required before fusion.

Fusions were performed with the non-immunoglobulin secreting Kearneyvariant of P3X63/Ag8.653 using our standard procedure as previouslypublished (Wikstrand et al, 1982; Wikstrand et al, 1986). Spleen cell:myeloma ratios ranged from 5-10:1, and spleen cell equivalent densitywas adjusted to 2-4×10⁵ cells/well for plating. Supernatants fromoutgrowing hybrids were screened as appropriate to the immunizationprotocol: Protocol 1, Pep 3 and D270 MG xenograft cells for positivity;all positive hybrids tested for nonreactivity on D270 MG cultured cells,which do not express type II mutant of EGFR, A431 cells to determinespecificity; Protocols 2 and 3, Pep 3 and HC for positivity, NIH Swiss3T3 (3T3; non-transfected parental cell line) for non-reactivity, A431cells to determine specificity; Protocol 4, HC Triton X-100 extractpreparation for positivity, A431 Triton X-100 extract preparation todetermine specificity.

Antibody Purification

Mabs were purified as previously described (Wikstrand et al, 1986; He etal, 1994), with the exception that Mab L8A4 was purified on a Protein Gcolumn (GammaBind Plus, Pharmacia). Rabbit anti-Pep 3 polyvalent serumwas purified on Pep 3 affinity columns as previously described (Humphreyet al, 1990, Wikstrand et al, 1993). The column was prepared by thecoupling of peptide to cyanogen bromide-activated Sepharose, serum waspassed over the column, and eluted with 100 mM glycine buffer (pH 3.0)with immediate neutralization with 1/10 volume 1M Hepes buffer (pH 8.0).

Immunohistochemical analysis of acetone fixed (−70° C., 30 seconds)tissue sections of human normal or tumor tissue, rat glioblastomaxenografts derived from human tumors or transfected cell lines, orcultured cells plated on LabTek slides was performed as previouslydescribed (Humphrey et al 1988; 1990). Primary antibody concentrationsused in these assays were 10 and 5 μg/ml for Mabs L8A4, Y10, and IgG1and IgG2a irrelevant controls, and 5 and 2.5 μg/ml for Mab 528 andpolyvalent Pep 3-affinity purified rabbit serum.

RT-PCR identification of type II mutant of EGFR mRNA

RNA was purified from 2×20 μm sections of frozen breast carcinoma orcontrol tissues using the guanidium isothiocyanate-acid phenol methodChomczynski and Sacchi, 1987). Tissue controls included the type IIMutant of EGFR expressing human glioma D256 MG, and the NR6W transfectedNIH Swiss 3T-3 cell line expressing normal EGFR; both were grown assubcutaneous xenografts in athymic rats. Additional controls includedthe cell lines NR6, NR6W, and NR6M, which express no EGFR, normal EGFR,and type II mutant of EGFR, respectively (Castelino-Prabhu et al, 1994).Reverse transcription and polymerase chain reaction were performed aspreviously described (Hale et al, 1995) except that forty cycles ofamplification were performed using the parameters 95° C. 80 sec. 52° C.1 min, 72° C. 2 min. Ten μl of each of the products were analyzed byelectrophoresis on 2.0% agarose gels in 0.5X TAE buffer (1×=0.04 MTris-acetate, 0.001 M EDTA) using 100 bp markers (BRL-GIBCO) as sizestandards, followed by ethidium bromide staining. Primers for PCR ofwild type EGFR and/or variants were forward5′GGGGAATTCGCGATGCGACCCTCCGGG3′; reverse5′GGGAAGCTTTCCGTTACACACTTTGCG3′. Using these primers, the sizes of theexpected normal and type II mutant of EGFR products are 1037 bp and 236bp, respectively.

The patents and literature articles cited in this disclosure areexpressly incorporated herein by reference.

What is claimed is:
 1. A method of immunizing a mammal, comprising thestep of: administering a type II EGFR mutant peptide to a mammal,whereby antibodies which are imunoreactive with epitopes found on thetype II EGFR mutant peptide but not found on normal EGFR are produced.2. The method of claim 1 wherein the mammal is a mouse.
 3. The method ofclaim 1 wherein the mammal is a rabbit.
 4. The method of claim 1 whereinthe mammal is a goat.
 5. The method of claim 1 wherein the mammal is arat.
 6. The method of claim 1 wherein the administering is subcutaneous.7. The method of claim 1 wherein the administering is intradermal. 8.The method of claim 1 wherein the administering is intravenous.
 9. Themethod of claim 1 wherein the administering is intraperitoneal.
 10. Themethod of claim 1 wherein the peptide is coupled to keyhole limpethemocyanin.
 11. The method of claim 1 wherein said peptide comprises anamino acid sequence LEEKKGNYVVT.
 12. The method of claim 1 wherein saidpeptide comprises an amino acid sequence LEEKKGNYVVTDHC.